Phytase enzymes, nucleic acid sequences encoding phytase enzymes and vectors and host cells incorporating same

ABSTRACT

DNA is provided which encodes an enzyme having phytase activity isolated from  Penicillium, Fusarium, Humicola  and  Emericella . Also provided for is a method of isolating DNA encoding an enzyme having phytase activity from organisms which possess such DNA, transformation of the DNA into a suitable host organism, expression of the transformed DNA and the use of the expressed phytase protein in feed as a supplement.

The present application is a divisional application of U.S. patentapplication Ser. No. 10/492,782, filed on Apr. 14, 2004, which is a 371of PCT/US02/34256, filed on Oct. 25, 2002, which claims priority to U.S.Provisional Patent Application Ser. No. 60/339,552, filed on Oct. 26,2001.

FIELD OF THE INVENTION

The present invention relates to phytase, nucleic acid sequencesencoding phytase, as well as is the production of phytase and its use.

REFERENCES

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BACKGROUND OF THE INVENTION

Phosphorous (P) is an essential element for growth. A substantial amountof the phosphorous found in conventional livestock feed, e.g., cerealgrains, oil seed meal, and by products that originate from seeds, is inthe form of phosphate which is covalently bound in a molecule know asphytate (myo-inositol hexakisphosphate). The bioavailability ofphosphorus in this form is generally quite low for non-ruminants, suchas poultry and swine, because they lack digestive enzymes for separatingphosphorus from the phytate molecule.

Several important consequences of the inability of non-ruminants toutilize phytate may be noted. For example, expense is incurred wheninorganic phosphorus (e.g., dicalcium phosphate, defluorinatedphosphate) or animal products (e.g., meat and bone meal, fish meal) areadded to meet the animals' nutritional requirements for phosphorus.Additionally, phytate can bind or chelate a number of minerals (e.g.,calcium, zinc, iron, magnesium, copper) in the gastrointestinal tract,thereby rendering them unavailable for absorption. Furthermore, most ofthe phytate present in feed passes through the gastrointestinal tract,elevating the amount of phosphorous in the manure. This leads to anincreased ecological phosphorous burden on the environment.

Ruminants, such as cattle, in contrast, readily utilize phytate thanksto an enzyme produced by rumen microorganisms known as phytase. Phytasecatalyzes the hydrolysis of phytate to (1) myo-inositol and/or (2)mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3)inorganic phosphate. Two different types of phytases are known: (1) aso-called 3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC3.1.3.8) and (2) a so-called 6-phytase (myo-inositol hexaphosphate6-phosphohydrolase, EC 3.1.3.26). The 3-phytase preferentiallyhydrolyzes first the ester bond at the 3-position, whereas the 6-phytasepreferentially hydrolyzes first the ester bond at the 6-position.

Microbial phytase, as a feed additive, has been found to improve thebioavailability of phytate phosphorous in typical non-ruminant diets(See, e.g., Cromwell, et al., 1993). The result is a decreased need toadd inorganic phosphorous to animal feeds, as well as lower phosphorouslevels in the excreted manure (See, e.g., Kornegay, et al., 1996).

Despite such advantages, few of the known phytases have gainedwidespread acceptance in the feed industry. The reasons for this varyfrom enzyme to enzyme. Typical concerns relate to high is manufacturecosts and/or poor stability/activity of the enzyme in the environment ofthe desired application (e.g., the pH/temperature encountered in theprocessing of feedstuffs, or in the digestive tracts of animals).

It is, thus, generally desirable to discover and develop novel enzymeshaving good stability and phytase activity for use in connection withanimal feed, and to apply advancements in fermentation technology to theproduction of such enzymes in order to make them commercially viable. Itis also desirable to ascertain nucleotide sequences which can be used toproduce more efficient genetically engineered organisms capable ofexpressing such phytases in quantities suitable for industrialproduction. It is still further desirable to develop a phytaseexpression system via genetic engineering which will enable thepurification and utilization of working quantities of relatively pureenzyme.

SUMMARY OF THE INVENTION

The present invention provides for a purified enzyme having phytaseactivity which is derived from a microbial source, and preferably from afungal source, such as, a Penicillium species, e.g., P. chrysogenum(deposit no. NRRL 1951), a Fusarium species, e.g. F. javanicum (depositno. CBS 203.32) or F. vertisillibodes, a Humicola species, e.g., H.grisea (deposit no. ATCC 22081 or CBS 225.63), or an Emericella species,e.g., E. desertorum (deposit no. CBS 653.73).

The present invention further provides a polynucleotide sequence codingfor the enzyme comprising a DNA as shown in FIG. 1(SEQ ID NO:1) or FIGS.19A-19C (SEQ ID NO:43); a polynucleotide which encodes the amino acidsequence shown in FIG. 2 (SEQ ID NO:2), 3 (SEQ ID NO:3) or 19A-19C (SEQID NO:44); a polynucleotide which encodes a phytase which comprises anamino acid segment which differs from the sequence in FIG. 2 (SEQ IDNO:2) or FIG. 3 (SEQ ID NO:3) or FIGS. 19A-19C (SEQ ID NO:44), providedthat the polynucleotide encodes a derivative of the phytase specificallydescribed herein; and a polynucleotide which encodes a phytase thatcomprises an amino acid sequence which differs from the sequence in FIG.2 (SEQ ID NO:2) or FIG. 3 (SEQ ID NO:3) or FIGS. 19A-19C (SEQ ID NO:44),provided that the polynucleotide hybridizes under medium to highstringency conditions with a nucleic acid sequence comprising all orpart of the nucleic acid sequence in FIG. 1 (SEQ ID NO:1) or FIGS.19A-19C (SEQ ID NO:43).

The present invention also provides a polynucleotide encoding an enzymehaving phytate hydrolyzing activity and including a nucleotide sequenceas shown in FIG. 4, 7, 18A-18C or 21 (SEQ ID NO:4, 7, 41 and 7,respectively); a polynucleotide which encodes the amino acid sequenceshown in FIG. 5, 6, 8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8,respectively); a polynucleotide which encodes a phytase which comprisesan amino acid segment which differs from the sequence in FIG. 5, 6, 8,18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8, respectively), providedthat the polynucleotide encodes a derivative of the phytase specificallydescribed herein; and a polynucleotide which encodes a phytase thatcomprises an amino acid segment which differs from the sequence in FIG.5, 6, 8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8, respectively),provided that the polynucleotide hybridizes under medium to highstringency conditions with a nucleotide sequence as shown in FIG. 4, 7,18A-18C or 21 (SEQ ID NO:4, 7,41 and 7, respectively).

The present invention further provides a polynucleotide encoding anenzyme having phytate hydrolyzing activity and including a nucleotidesequence as shown in FIG. 9 or FIGS. 20A-20C (SEQ ID NO:9 or 45,respectively); a polynucleotide which encodes the amino acid sequenceshown in FIG. 10, 11, or 20A-20B (SEQ ID NO:10, 11, and 46,respectively); a polynucleotide which encodes a phytase which comprisesan amino acid segment which differs from the sequence in FIG. 10, 11, or20A-20B (SEQ ID NO:10, 11, and 46, respectively), provided that thepolynucleotide encodes a derivative of the phytase specificallydescribed herein; and a polynucleotide which encodes a phytase thatcomprises an amino acid segment which differs from the sequence in FIG.10, 11, or 20A-20B (SEQ ID NO:10, 11, and 46, respectively), providedthat the polynucleotide hybridizes under medium to high stringencyconditions with a nucleotide sequence as shown in FIG. 9 or FIGS.20A-20C (SEQ ID NO:9 or 45, respectively).

The present invention further provides a polynucleotide encoding anenzyme having phytate hydrolyzing activity and including a nucleotidesequence as shown in FIG. 17A (SEQ ID NO:39); a polynucleotide whichencodes the amino acid sequence shown in FIG. 17B (SEQ ID NO:40); apolynucleotide which encodes a phytase which comprises an amino acidsegment which differs from the sequence in FIG. 17B (SEQ ID NO:40),provided that the polynucleotide encodes a derivative of the phytasespecifically described herein; and a polynucleotide which encodes aphytase that comprises an amino acid segment which differs from thesequence in FIG. 17B (SEQ ID NO:40), provided that the polynucleotidehybridizes under medium to high stringency conditions with a nucleotidesequence as shown in FIG. 17A (SEQ ID NO:39).

Additionally, the present invention encompasses vectors which includethe polynucleotide sequences described above, host cells which have beentransformed with such polynucleotides or vectors, fermentation brothscomprising such host cells and phytase proteins encoded by suchpolynucleotides which are expressed by the host cells. Preferably, thepolynucleotide of the invention is in purified or isolated form and isused to prepare a transformed host cell capable of producing the encodedprotein product thereof. Additionally, polypeptides which are theexpression product of the polynucleotide sequences described above arewithin the scope of the present invention.

In one embodiment, the present invention provides an isolated orpurified polynucleotide derived from a fungal source of the genusPenicillium, which polynucleotide comprises a nucleotide is sequenceencoding an enzyme having phytase activity. The fungal source can be,for example, from Penicillium chrysogenum. In another embodiment, theinvention provides an isolated or purified polynucleotide derived from afungal source of the genus Fusarium, which polynucleotide comprises anucleotide sequence encoding an enzyme having phytase activity. Thefungal source can be selected, for example, from the group consisting ofFusarium javanicum and Fusarium vertisillibodes. In yet anotherembodiment, the present invention provides an isolated or purifiedpolynucleotide derived from a fungal source of the genus Humicola, whichpolynucleotide comprises a nucleotide sequence encoding an enzyme havingphytase activity. The fungal source can be, for example, from Humicolagrisea. In still another embodiment, the present invention provides anisolated or purified polynucleotide derived from a fungal source of thegenus Emericella, which polynucleotide comprises a nucleotide sequenceencoding an enzyme having phytase activity. The fungal source can be,for example, from Emericella desertorum.

According to one embodiment, the polynucleotide encodes aphytate-hydrolyzing enzyme including an amino acid sequence having atleast 55% identity, preferably at least 60% identity, more preferably atleast 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to an amino acid sequence as disclosed in FIG. 2, 3or 19A-19C (SEQ ID NO:2, 3, and 44, respectively).

One embodiment of the present invention provides an isolatedpolynucleotide comprising a nucleotide sequence (i) having at least 55%identity, preferably at least 60% identity, more preferably at least 65%identity, still more preferably at least 70% identity, yet morepreferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 1 or19A-19C (SEQ ID NO:1 and 43, respectively), or (ii) being capable ofhybridizing to a probe derived from the nucleotide sequence disclosed inFIG. 1 or 19A-19C (SEQ ID NO:1 and 43, respectively) under conditions ofintermediate to high stringency, or (iii) being complementary to thenucleotide sequence disclosed in FIG. 1 or 19A-19C (SEQ ID NO:1 and 43,respectively).

Another aspect of the present invention provides an isolatedpolynucleotide encoding an enzyme having phytase activity, wherein theenzyme is derived from a Penicillium source. The source can be, forexample, Penicillium chrysogenum.

In one embodiment, the polynucleotide encodes a phytate-hydrolyzingenzyme that includes an amino acid sequence having at least 55%identity, preferably at least 60% identity, more preferably at least 65%identity, still more preferably at least 70% identity, yet morepreferably at least 75% is identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to an amino acid sequence as disclosed in FIG. 5, 6,8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8, respectively).

In another embodiment, the polynucleotide encoding a phytate-hydrolyzingenzyme has at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to a nucleotidesequence as disclosed in FIG. 1 or 19A-19C (SEQ ID NO:1 and 43,respectively), or (ii) is capable of hybridizing to a probe derived fromthe nucleotide sequence disclosed in FIG. 1 or 19A-19C (SEQ ID NO:1 and43, respectively) under conditions of medium to high stringency, or(iii) is complementary to the nucleotide sequence disclosed in FIG. 1 or19A-19C (SEQ ID NO:1 and 43, respectively).

Yet a further aspect of the present invention provides an expressionconstruct including a polynucleotide sequence (i) having at least 55%identity, preferably at least 60% identity, more preferably at least 65%identity, still more preferably at least 70% identity, yet morepreferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 1 or19A-19C (SEQ ID NO:1 and 43, respectively), or (ii) being capable ofhybridizing to a probe derived from the nucleotide sequence disclosed inFIG. 1 or 19A-19C (SEQ ID NO:1 and 43, respectively) under conditions ofmedium to high stringency, or (iii) being complementary to thenucleotide sequence disclosed in FIG. 1 or 19A-19C (SEQ ID NO:1 and 43,respectively). Also provided are a vector (e.g., a plasmid) includingsuch expression construct, and a host cell (such as an Aspergillus,e.g., Aspergillus niger or Aspergillus nidulans) transformed with such avector.

In another of its aspects, the present invention provides a probe foruse in detecting nucleic acid sequences coding for an enzyme havingphytase activity derived from a microbial source, comprising: anucleotide sequence (i) having at least 55% identity, preferably atleast 60% identity, more preferably at least 65% identity, still morepreferably at least 70% identity, yet more preferably at least 75%identity, even more preferably at least 80% identity, again morepreferably at least 85% identity, yet again more preferably at least 90%identity, and most preferably at least 95% up to about 100% identity toa nucleotide sequence as disclosed in FIG. 1 or 19A-19C (SEQ ID NO:1 and43, respectively), or (ii) being capable of hybridizing to apolynucleotide including a sequence as disclosed in FIG. 1 or 19A-19C(SEQ ID NO:1 and 43, respectively) under conditions of medium to highstringency, or (iii) being complementary to the nucleotide sequencedisclosed in FIG. 1 or 19A-19C (SEQ ID NO:1 and 43, respectively).

In one embodiment, the microbial source is a fungal source, e.g., aPenicillium species, such as Penicillium chrysogenum.

The present invention additionally provides a food or animal feedincluding an enzyme having phytase activity, wherein the enzymecomprises an amino acid sequence having at least 55% identity,preferably at least 60% identity, more preferably at least 65% identity,still more preferably at least 70% identity, yet more preferably atleast 75% identity, even more preferably at least 80% identity, againmore preferably at least 85% identity, yet again more preferably atleast 90% identity, and most preferably at least 95% up to about 100%identity to an amino acid sequence as disclosed in FIG. 2, 3 or 19A-19C(SEQ ID NO:2, 3, and 44, respectively).

The present invention provides food or animal feed including an enzymehaving phytase activity, wherein the enzyme is derived from a fungalsource such as Penicillium chrysogenum.

Another aspect of the present invention provides a method of producingan enzyme having phytase activity, comprising:

-   -   (a) providing a host cell transformed with an expression vector        comprising a polynucleotide as described herein;    -   (b) cultivating the transformed host cell under conditions        suitable for the host cell to produce the phytase; and    -   (c) recovering the phytase.

According to one embodiment, the host cell is an Aspergillus species,such as A. niger or A. nidulans.

In one embodiment, the host cell is a plant cell. In this embodiment,cells or entire transformed plants may be grown and used.

Another aspect of the present invention provides a method of producingan enzyme having phytase activity, comprising:

-   -   (a) providing a host cell transformed with an expression vector        comprising a polynucleotide as described herein;    -   (b) cultivating the transformed host cell under conditions        suitable for the host cell to produce the phytase. The        transformed cells, as well as organisms grown from such cells,        may be used without further isolation of the enzyme.

In another aspect, the invention provides a purified enzyme havingphytase activity, produced by the methods described above.

In yet another of its aspects, the present invention provides a methodof separating phosphorous from phytate, comprising the step of treatingthe phytate with an enzyme comprising an amino acid sequence having atleast 55% identity, preferably at least 60% identity, more preferably atleast 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to an amino acid sequence as disclosed in FIG. 2, 3or 19A-19C (SEQ ID NO:2, 3, and 44, respectively).

The present invention further provides a method of separatingphosphorous from phytate, comprising the step of treating the phytatewith an enzyme as defined above.

Another aspect of the present invention provides a phytate-hydrolyzingenzyme that includes an amino acid sequence having at least 55%identity, preferably at least 60% identity, more preferably at least 65%identity, still more preferably at least 70% identity, yet morepreferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to an amino acid sequence as disclosed in FIG. 5, 6,8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8, respectively). In adifferent embodiment, the sequence compares in the same way to thesequence as disclosed in FIG. 10, 11 or 20A-20B (SEQ ID NO:10, 11, and46, respectively).

A further aspect of the present invention provides an isolatedpolynucleotide including a nucleotide sequence (i) having at least 55%identity, preferably at least 60% identity, more preferably at least 65%identity, still more preferably at least 70% identity, yet morepreferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 5, 6,8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8, respectively), or (ii)being capable of hybridizing to a probe derived from the nucleotidesequence disclosed in FIG. 5, 6, 8, 18A-18C or 21 (SEQ ID NO:5, 6, 8,42, and 8, respectively) under conditions of intermediate to highstringency, or (iii) being complementary to the nucleotide sequencedisclosed in FIG. 5, 6, 8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8,respectively).

In another embodiment, the invention provides an isolated polynucleotideincluding a nucleotide sequence (i) having at least 55% identity,preferably at least 60% identity, more preferably at least 65% identity,still more preferably at least 70% identity, yet more preferably atleast 75% is identity, even more preferably at least 80% identity, againmore preferably at least 85% identity, yet again more preferably atleast 90% identity, and most preferably at least 95% up to about 100%identity to a nucleotide sequence as disclosed in FIG. 10, 11 or 20A-20B(SEQ ID NO:10, 11, and 46, respectively), or (ii) being capable ofhybridizing to a probe derived from the nucleotide sequence disclosed inFIG. 10, 11 or 20A-20B (SEQ ID NO:10, 11, and 46, respectively) underconditions of intermediate to high stringency, or (iii) beingcomplementary to the nucleotide sequence disclosed in FIG. 10, 11 or20A-20B (SEQ ID NO:10,11, and 46, respectively).

In one embodiment, the isolated polynucleotide encodes aphytate-hydrolyzing enzyme derived from a member of the Fusarium genus,preferably from F. javanicum or F. vertisillibodes. The enzyme includes,according to one embodiment, an amino acid sequence having at least 55%identity, preferably at least 60% identity, more preferably at least 65%identity, still more preferably at least 70% identity, yet morepreferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to an amino acid sequence as disclosed in FIG. 5, 6,8, 18A-18C or 21 (SEQ ID NO:5, 6, 8, 42, and 8, respectively).

In a different embodiment, the isolated polynucleotide encodes aphytate-hydrolyzing enzyme derived from a member of the Humicola genus,preferably from H. grisea. The enzyme includes, according to oneembodiment, an amino acid sequence having at least 55% identity,preferably at least 60% identity, more preferably at least 65% identity,still more preferably at least 70% identity, yet more preferably atleast 75% identity, even more preferably at least 80% identity, againmore preferably at least 85% identity, yet again more preferably atleast 90% identity, and most preferably at least 95% up to about 100%identity to an amino acid sequence as disclosed in FIG. 10, 11 or20A-20B (SEQ ID NO:10, 11, and 46, respectively).

In still another embodiment, the isolated polynucleotide encodes aphytate-hydrolyzing enzyme derived from a member of the Emericellagenus, preferably from E. desertorum. The enzyme includes, according toone embodiment, an amino acid sequence having at least 55% identity,preferably at least 60% identity, more preferably at least 65% identity,still more preferably at least 70% identity, yet more preferably atleast 75% identity, even more preferably at least 80% identity, againmore preferably at least 85% identity, yet again more preferably atleast 90% identity, and most preferably at least 95% up to about 100%identity to an amino acid sequence as disclosed in FIG. 17B (SEQ IDNO:40).

In another embodiment, the polynucleotide encoding a phytate-hydrolyzingenzyme includes a nucleotide sequence (i) having at least 55% identity,preferably at least 60% identity, more preferably at least 65% identity,still more preferably at least 70% identity, yet more preferably atleast 75% identity, even more preferably at least 80% identity, againmore preferably at least 85% identity, yet again more preferably atleast 90% identity, and most preferably at least 95% up to about 100%identity to a nucleotide sequence as disclosed in FIG. 4, 7, 18A-18C,and 21 (SEQ ID NO:4, 7, 41, and 7, respectively), or (ii) capable ofhybridizing to a probe derived from the nucleotide sequence disclosed inFIG. 4, 7, 18A-18C, and 21 (SEQ ID NO:4, 7,41, and 7, respectively)under conditions of medium to high stringency, or (iii) complementary tothe nucleotide sequence disclosed in FIG. 4, 7, 18A-18C, and 21 (SEQ IDNO:4, 7, 41, and 7, respectively).

In still another embodiment, the polynucleotide encoding aphytate-hydrolyzing enzyme includes a nucleotide sequence (i) having atleast 55% identity, preferably at least 60% identity, more preferably atleast 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 9 or20A-20C (SEQ ID NO:9 and 45, respectively), or (ii) capable ofhybridizing to a probe derived from the nucleotide sequence disclosed inFIG. 9 or 20A-20C (SEQ ID NO:9 and 45, respectively) under conditions ofmedium to high stringency, or (iii) complementary to the nucleotidesequence disclosed in FIG. 9 or 20A-20C (SEQ ID NO:9 and 45,respectively).

In still another embodiment, the polynucleotide encoding aphytate-hydrolyzing enzyme includes a nucleotide sequence (i) having atleast 55% identity, preferably at least 60% identity, more preferably atleast 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 17A(SEQ ID NO:39), or (ii) capable of hybridizing to a probe derived fromthe nucleotide sequence disclosed in FIG. 17A (SEQ ID NO:39) underconditions of medium to high stringency, or (iii) complementary to thenucleotide sequence disclosed in FIG. 17A (SEQ ID NO:39).

Another aspect of the present invention provides an expression constructcomprising a polynucleotide including a nucleotide sequence (i) havingat least 55% identity, preferably at least 60% identity, more preferablyat least 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 4, 7,18A-18C, and 21 (SEQ ID NO:4, 7, 41, and 7, respectively), or (ii) beingcapable of hybridizing to a probe derived from the nucleotide sequencedisclosed in FIG. 4, 7, 18A-18C, and 21 (SEQ ID NO:4, 7, 41, and 7,respectively) under conditions of medium to high stringency, or (iii)being complementary to the nucleotide sequence disclosed in FIG. 4, 7,18A-18C, and 21 (SEQ ID NO:4, 7, 41, and 7, respectively).

Alternatively, the present invention provides an expression constructcomprising a polynucleotide including a nucleotide sequence (i) havingat least 55% identity, preferably at least 60% identity, more preferablyat least 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to a nucleotide sequence as disclosed in FIG. 9 or20A-20C (SEQ ID NO:9 and 45, respectively), or (ii) being capable ofhybridizing to a probe derived from the nucleotide sequence disclosed inFIG. 9 or 20A-20C (SEQ ID NO:9 and 45, respectively) under conditions ofmedium to high stringency, or (iii) being complementary to thenucleotide sequence disclosed in FIG. 9 or 20A-20C (SEQ ID NO:9 and 45,respectively).

In another embodiment, the present invention provides an expressionconstruct comprising a polynucleotide including a nucleotide sequence(i) having at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to a nucleotidesequence as disclosed in FIG. 17A (SEQ ID NO:39), or (ii) being capableof hybridizing to a probe derived from the nucleotide sequence disclosedin FIG. 17A (SEQ ID NO:39) under conditions of medium to highstringency, or (iii) being complementary to the nucleotide sequencedisclosed in FIG. 17A (SEQ ID NO:39).

The present invention further provides a vector (e.g., plasmid)including such an expression construct, as well as a host cell (e.g.,Aspergillus niger or Aspergillus nidulans) transformed with a vector asdescribed above.

The present invention additionally provides a probe for use in detectingnucleic acid sequences coding for an enzyme having phytase activityderived from a microbial source, comprising: a nucleotide sequence (i)having at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to a nucleotidesequence as disclosed in FIG. 4, 7, 18A-18C, and 21 (SEQ ID NO:4, 7, 41,and 7, respectively), or (ii) being capable of hybridizing to apolynucleotide including a sequence as disclosed in FIG. 4, 7, 18A-18C,and 21 (SEQ ID NO:4, 7, 41, and 7, respectively) under conditions ofmedium to high stringency, or (iii) being complementary to thenucleotide sequence disclosed in FIG. 4, 7,18A-18C, and 21 (SEQ ID NO:4,7, 41, and 7, respectively).

In another aspect, the invention provides a probe for use in detectingnucleic acid sequences coding for an enzyme having phytase activityderived from a microbial source, comprising: a nucleotide sequence (i)having at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to a nucleotidesequence as disclosed in FIG. 9 or 20A-20C (SEQ ID NO:9 and 45,respectively), or (ii) being capable of hybridizing to a polynucleotideincluding a sequence as disclosed in FIG. 9 or 20A-20C (SEQ ID NO:9 and45, respectively) under conditions of medium to high stringency, or(iii) being complementary to the nucleotide sequence disclosed in FIG. 9or 20A-20C (SEQ ID NO:9 and 45, respectively).

In another aspect, the invention provides a probe for use in detectingnucleic acid sequences coding for an enzyme having phytase activityderived from a microbial source, comprising: a nucleotide sequence (i)having at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to a nucleotidesequence as disclosed in FIG. 17A (SEQ ID NO:39), or (ii) being capableof hybridizing to a polynucleotide including a sequence as disclosed inFIG. 17A (SEQ ID NO:39) under conditions of medium to high stringency,or (iii) being complementary to the nucleotide sequence disclosed inFIG. 17A (SEQ ID NO:39).

In one embodiment, the microbial source is a fungal source, e.g., aPenicillium species, such as P. chrysogenum, a Fusarium species, such asF. javanicum or F. vertisillibodes, an Emericella species such as E.desertorum or a Humicola species, such as H. grisea.

The present invention further provides a food or animal feed includingan enzyme having phytase activity, wherein the enzyme includes an aminoacid sequence having at least 55% identity, preferably at least 60%identity, more preferably at least 65% identity, still more preferablyat least 70% identity, yet more preferably at least 75% identity, evenmore preferably at least 80% identity, again more preferably at least85% identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to an amino acidsequence as disclosed in FIG. 5, 6, 8, 10, 11, 17B, 18A-18C, 19A-19C,20A-20B or 21 (SEQ ID NO:5, 6, 8, 10, 11, 40, 42, 44, and 46,respectively).

Still further, the present invention provides a method of separatingphosphorous from phytate, comprising the step of treating the phytatewith an enzyme (i) having phytate hydrolyzing activity and (ii)including an amino acid sequence having at least 55% identity,preferably at least 60% identity, more preferably at least 65% identity,still more preferably at least 70% identity, yet more preferably atleast 75% identity, even more preferably at least 80% identity, againmore preferably at least 85% identity, yet again more preferably atleast 90% identity, and most preferably at least 95% up to about 100%identity to an amino acid sequence as disclosed in FIG. 5, 6, 8, 18A-18Cor 21 (SEQ ID NO:5, 6, 8, 42 and 8, respectively). In another aspect,the invention provides a method of separating phosphorous from phytate,comprising the step of treating the phytate with an enzyme (i) havingphytate hydrolyzing activity and (ii) including an amino acid sequencehaving at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to an amino acidsequence as disclosed in FIG. 10, 11 or 20A-20B (SEQ ID NO:10, 11, and46, respectively), or as disclosed in FIG. 17B (SEQ ID NO:40).

As will be appreciated, an advantage of the present invention is that apolynucleotide has been isolated which provides the capability ofisolating further polynucleotides which encode proteins having phytaseactivity.

Another advantage of the present invention is that, by virtue ofproviding a polynucleotide encoding a protein having phytase activity,it is possible to produce, through recombinant means, a host cell whichis capable of producing the protein having phytase activity inrelatively large quantities.

Yet another advantage of the present invention is that commercialapplication of proteins having phytase activity is made practical. Forexample, the present invention provides animal feed incorporating thephytase described herein.

Other objects and advantages of the present invention will becomeapparent from the following detailed specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleic acid sequence (SEQ ID NO:1) corresponding to 1317base pairs of a gene encoding a phytase hydrolyzing enzyme derived fromPenicillium chrysogenum.

FIG. 2 shows an amino acid sequence (SEQ ID NO:2) of a phytase enzymeencoded by the nucleic acid sequence of FIG. 1 (SEQ ID NO:1).

FIG. 3 shows an amino acid sequence (SEQ ID NO:3) of a mature chimericphytase enzyme, produced via the expression of a nucleic acid sequenceencoding an Aspergillus signal sequence, linked to a nucleic acidsequence encoding a phytase hydrolyzing enzyme derived from Penicilliumchrysogenum.

FIG. 4 shows a nucleic acid sequence (SEQ ID NO:4) corresponding to 1299base pairs of a gene encoding a phytase hydrolyzing enzyme derived fromFusarium javanicum.

FIG. 5 shows an amino acid sequence (SEQ ID NO:5) of a phytase enzymeencoded by the nucleic acid sequence of FIG. 4 (SEQ ID NO:4).

FIG. 6 shows an amino acid sequence (SEQ ID NO:6) of a mature chimericphytase enzyme, produced via the expression of a nucleic acid sequenceencoding an Aspergillus signal sequence, linked to a nucleic acidsequence encoding a phytase hydrolyzing enzyme derived from Fusariumjavanicum.

FIG. 7 shows a nucleic acid sequence (SEQ ID NO:7) corresponding to 224base pairs of a gene encoding a phytase hydrolyzing enzyme derived fromFusarium vertisillibodes.

FIG. 8 shows an amino acid sequence (SEQ ID NO:8) of a phytase enzymeencoded by the nucleic acid sequence of FIG. 7 (SEQ ID NO:7).

FIG. 9 shows a nucleic acid sequence (SEQ ID NO:9) corresponding to 224base pairs of a gene encoding a phytase hydrolyzing enzyme derived fromHumicola grisea.

FIG. 10 shows an amino acid sequence (SEQ ID NO:10) of a phytase enzymeencoded by the nucleic acid sequence of FIG. 9 (SEQ ID NO:9).

FIG. 11 shows an amino acid sequence (SEQ ID NO:11) of a mature chimericphytase enzyme, produced via the expression of a nucleic acid sequenceencoding an Aspergillus signal sequence, linked to a nucleic acidsequence encoding a phytase hydrolyzing enzyme derived from Humicolagrisea.

FIG. 12 shows a nucleic acid sequence (SEQ ID NO:12) corresponding to192 base pairs of a gene fragment encoding the amino end, including asignal sequence, for a phytase hydrolyzing enzyme derived fromAspergillus niger. This sequence includes an ATG start codon at the 5□end and an intron extending from residues 46 to 147.

FIG. 13 shows an amino acid sequence (SEQ ID NO:13) of the amino end,including a signal sequence, of a phytase enzyme encoded by the nucleicacid sequence of FIG. 12 (SEQ ID NO:12).

FIGS. 14A-14D show alignments of amino acid sequences disclosed hereinwith published amino acid sequences of known phytase enzymes. FIG. 14Ashows a GAP alignment, as further described below, of the amino acidsequence of a mature (i.e., lacking the signal sequence corresponding toamino acids 1-23) phytase from A. niger (accession number P34752, 444amino acids; top row of each pair, SEQ ID NO:14) and a phytase derivedfrom P. chrysogenum (FIG. 3, 446 amino acids; bottom row of each pair,SEQ ID NO:15). Straight lines between the aligned sequences indicateidentical residues, dots between the aligned sequences indicate similarresidues. The two sequences show 65% identity, 70% similarity.

FIG. 14B shows a BLAST alignment (TBLASTN 2.0.5 program, SEQ ID NO:18),as further described below, of residues 1-445 of the amino acid sequence(SEQ ID NO:16) from P. chrysogenum (P.c.) disclosed in FIG. 3 and theamino acid sequence determined from nucleic acid residues 407 to 1732 ofa cDNA sequence encoding an Aspergillus fumigatus (A.f.) phytase(accession number U59804, SEQ ID NO:17). Letters between the alignedsequences indicate identical amino acid residues, pluses indicatesimilar residues. These portions of the two sequences show 62% identity,75% similarity.

FIG. 14C shows a BLAST alignment of (SEQ ID NO:21) residues 4-445 of theamino acid sequence (SEQ ID NO:19) from P. chrysogenum (P.c.) disclosedin FIG. 3 and the amino acid sequence determined from nucleic acidresidues 411 to 1730 of a cDNA sequence encoding an Aspergillus terreus(A.t.) phytase (accession number U60412, SEQ ID NO:20). These portionsof the two sequences show 60% identity, 73% similarity.

FIG. 14D shows a BLAST alignment (SEQ ID NO:24) of residues 7-445 of theamino acid sequence (SEQ ID NO:22) from P. chrysogenum (P.c.) disclosedin FIG. 3 and the amino acid sequence determined from nucleic acidresidues 293 to 1594 of a cDNA sequence encoding an Emericella nidulans(Aspergillus nidulans; A.t.) phytase (accession number U59803, SEQ IDNO:23). These portions of the two sequences show 60% identity, 75%similarity.

FIGS. 15A-15C show alignments of amino acid sequences disclosed hereinwith published amino acid sequences of known phytase enzymes. FIG. 15Ashows a GAP alignment of the 444 amino acid sequence of a mature phytasefrom A. niger (accession number P34752, SEQ ID NO:25) and a 440 aminoacid phytase sequence derived from F. javanicum (disclosed in FIG. 6,SEQ ID NO:26). The two sequences show 50% identity, 56% similarity.

FIG. 15B shows a GAP alignment of a 440 amino acid phytase sequencederived from F. javanicum (disclosed in FIG. 6, SEQ ID NO:27) and the463 amino acid sequence of a phytase from Emericella nidulans (E.n.)(Aspergillus nidulans; accession number U59803, SEQ ID NO:28). The twosequences show 52% identity, 60% similarity.

FIG. 15C shows a BLAST alignment (SEQ ID NO:31) of residues 7-438 of aphytase amino acid sequence (SEQ ID NO:29) from F. javanicum (F.j.)disclosed in FIG. 6 and the amino acid sequence (SEQ ID NO:30)determined from nucleic acid residues 2379 to 3719 of a cDNA sequenceencoding an Myceliopthora thermophila (M.t.) phytase (accession numberU59806). These portions of the two sequences show 52% identity, 68%similarity.

FIGS. 16A-16C show alignments of amino acid sequences disclosed hereinwith published amino acid sequences of known phytase enzymes. FIG. 16Ashows a GAP alignment of a 487 amino acid sequence of a phytase from M.thermophila (accession number U59806, SEQ ID NO:33) and a 449 amino acidphytase sequence derived from H. grisea (disclosed in FIG. 11, SEQ IDNO:32). The two sequences show 66% identity, 72% similarity.

FIG. 16B shows a GAP alignment of a 449 amino acid phytase sequencederived from H. grisea (disclosed in FIG. 11, SEQ ID NO:35) and the 444amino acid sequence of a mature phytase from A. niger (accession numberP34752, SEQ ID NO:34). The two sequences show 51% identity, 59%similarity.

FIG. 16C shows a BLAST alignment (SEQ ID NO:38) of residues 8-448 of aphytase amino acid sequence from H. grisea (H.g) (SEQ ID NO:36)disclosed in FIG. 11 and the amino acid sequence determined from nucleicacid residues 2340 to 3722 of a cDNA sequence encoding an Myceliopthorathermophila (M.t.) phytase (accession number U59806, SEQ ID NO:37).These portions of the two sequences show 65% identity, 74% similarity.

FIGS. 17A and 17B show the DNA encoding and amino acid sequence of aphytase from E. desertorum. FIG. 17A shows the sequence of genomic DNA(SEQ ID NO:39) encoding the gene for the phytase. Lower case letteringdepicts a putative intron. FIG. 17B shows the putative amino acidsequence (SEQ ID NO:40) encoded by the E desertorum phytase gene.

FIGS. 18A-18C show the genomic DNA sequence (SEQ ID NO:41) encoding aphytase from F. javanicum. The putative amino acid sequence (SEQ IDNO:42) of the phytase is indicated below the DNA sequence. A putativeintron is indicated below the DNA sequence by a horizontal line. Boxarrows below the DNA sequence indicate sequences of primers useful foramplifying the gene. Restriction sites are indicated above the sequencein bold.

FIGS. 19A-19C show the genomic DNA sequence (SEQ ID NO:43) encoding aphytase from P. chrysogenum. The putative amino acid sequence (SEQ IDNO:44) of the phytase is indicated below the DNA sequence. A putativeintron is indicated below the DNA sequence by a horizontal box. Boxarrows below the DNA sequence indicate sequences of primers useful foramplifying the gene. Restriction sites are indicated above the sequencein bold.

FIGS. 20A-20C show the genomic DNA sequence (SEQ ID NO:45) encoding aphytase from H. grisea. The putative amino acid sequence (SEQ ID NO:46)of the phytase is indicated below the DNA sequence. A putative intron isindicated below the DNA sequence by a horizontal line. Restriction sitesare indicated above the sequence in bold.

FIG. 21 shows a partial genomic DNA sequence (SEQ ID NO:7) encoding aphytase from F. vertisillibodes. The putative amino acid sequence (SEQID NO:8) of the phytase is indicated below the DNA sequence. A putativeintron is indicated below the DNA sequence by a horizontal line.Restriction sites are indicated above the sequence in bold.

FIG. 22 shows the DNA sequence (SEQ ID NO:47) of a gene encoding aphytase from E desertorum obtained using the procedure described inExample 1. Below the DNA sequence is indicated the putative amino acidsequence (SEQ ID NO:48) of the phytase. The arrows above the DNAsequence indicate ligation sequences (primers GSP1 rev:fyt037 andGSP2rev:fyt036) used to obtain upstream sequences of the gene (see FIG.17A).

FIG. 23 shows the DNA sequence (SEQ ID NO:49) of a gene encoding aphytase from F. javanicum obtained using the procedure described inExample 1. The putative amino acid sequence (SEQ ID NO:50) of thephytase is indicated below the DNA sequence. The arrows above the DNAsequence indicate ligation sequences (primers GSP1rev:fyt039 andGSP2rev:fyt038) used to obtain upstream sequences of the gene (see FIGS.18A-18B).

FIG. 24 shows results of expression of recombinantly produced phytasedescribed herein. FIG. 24A shows an isoelectric focusing (IEF) gelstained with Comassie blue. This gel sows protein present in thesupernatant from cultures of Aspergillus niger which had beentransformed with a vector encoding chimeric phytase from P. chrysogenum(lanes 5-8) and F. javanicum (lanes 9-12), as described in Examples 3and 4. The transformed host cells were grown under conditions designedto facilitate expression of the proteins encoded in the expressionvector. Lanes 1-3 (as marked) have nothing in them. Lane 4 hasfermentation broth from an A. nigertrans formed with the same vector asused for the F. javanicum and P. chrysogenum phytases, but comprising anucleic acid sequence encoding the native A. niger phytase enzyme. Lanes5-8 have fermentation broth from four different clones transformed withvector comprising the P. chrysogenum chymeric phytase, selected fortheir apparent high (lanes 5 and 8), moderate (lane 7) and low (lane 6)phytase activity, as determined in a preliminary test. Lanes 9-12 havefermentation broth from four different clones transformed with vectorcomprising the F. javanicum chymeric phytase, selected for theirapparent high (lane 11), moderate (lanes 9 and 10) and low (lane 12)phytase activity, as determined in a preliminary test. The Comassiestained gel indicates novel protein bands corresponding to phytaseactivity, as shown in the zymogram described in FIG. 24B, for each ofthe transformant types and no such novel protein bands for clones inwhich no activity was found.

FIG. 24B shows a zymogram produced as an overlay of the IEF geldescribed in FIG. 24A, made prior to staining of the gel, showing thephosphatase activity of the proteins in the gel. The zymogram indicatesphytase activity associated with the novel Comassie stained bands fromthe hosts transformed with chimeric phytase.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acid sequences are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. The headings provided herein are notlimitations of the various aspects or embodiments of the invention whichcan be had by reference to the specification as a whole. Accordingly,the terms defined immediately below are more fully defined by referenceto the specification as a whole.

“Protein”, as used herein, includes proteins, polypeptides, andpeptides. As will be appreciated by those in the art, the nucleic acidsequences of the invention, as defined below and further describedherein, can be used to generate protein sequences.

As used herein, the term “phytase” or “phytase activity” refers to aprotein or polypeptide which is capable of catalyzing the hydrolysis ofphytate to (1) myo-inositol and/or (2) mono-, di-, tri-, tetra- and/orpenta-phosphates thereof and (3) inorganic phosphate. For example,enzymes having catalytic activity as defined in Enzyme Commission ECnumber 3.1.3.8, or EC number 3.1.3.26.

In the broadest sense, by “nucleic acid sequence”, “polynucleotide” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid sequence of thepresent invention will generally contain phosphodiester bonds, althoughin some cases, as outlined below, nucleic acid sequence analogs areincluded that may have alternate backbones, comprising, for example,phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989)), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within one definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done for a varietyof reasons, for example to increase the stability and half-life of suchmolecules in physiological or food processing environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C.Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration. In addition, PNAs are not degraded by cellular enzymes,and thus can be more stable.

The nucleic acid sequences may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. As will be appreciated by those in the art, thedepiction of a single strand (“Watson”) also defines the sequence of theother strand (“Crick”); thus the sequences described herein alsoincludes the complement of the sequence. The nucleic acid sequence maybe DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acidsequence contains any combination of deoxyribo- and ribo-nucleotides,and any combination of bases, including uracil, adenine, thymine,cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,isoguanine, etc. As used herein, the term “nucleoside” includesnucleotides and nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide nucleic acid sequence,each containing a base, are referred to herein as a nucleoside.

The term “identical” in the context of two nucleic acid sequences orpolypeptide sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence, as measured usingone of the following sequence comparison or analysis algorithms.

“Optimal alignment” is defined as an alignment giving the highestpercent identity score. Such alignment can be performed using a varietyof commercially available sequence analysis programs, such as the localalignment program LALIGN using a ktup of 1, default parameters and thedefault PAM. A preferred alignment is the pairwise alignment performedusing the CLUSTAL-W program in MACVECTOR, operated in “slow” alignmentmode using default parameters, including an open gap penalty of 10.0, anextend gap penalty of 0.1, and a BLOSUM30 similarity matrix. If a gapneeds to be inserted into a first sequence to optimally align it with asecond sequence, the percent identity is calculated using only theresidues that are paired with a corresponding amino acid residue (i.e.,the calculation does not consider residues in the second sequences thatare in the “gap” of the first sequence).

Optimal alignment of sequences for comparison can also be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection.

“Percent sequence identity”, with respect to two amino acid orpolynucleotide sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 80% amino acid sequence identity means that 80% of the amino acidsin two optimally aligned polypeptide sequences are identical. Percentidentity can be determined, for example, by a direct comparison of thesequence information between two molecules by aligning the sequences,counting the exact number of matches between the two aligned sequences,dividing by the length of the shorter sequence, and multiplying theresult by 100. Readily available computer programs can be used to aid inthe analysis, such as ALIGN, Dayhoff, M. O. in “Atlas of ProteinSequence and Structure”, M. O. Dayhoff ed.,* 5 Suppl. 3:353-358,National Biomedical Research Foundation, Washington, D.C., which adaptsthe local homology algorithm of Smith and Waterman (1981) Advances inAppl. Math. 2:482-489 for peptide analysis. Programs for determiningnucleotide sequence identity are available in the Wisconsin SequenceAnalysis Package, Version 8 (available from Genetics Computer Group,Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, whichalso rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above.

An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol. 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. These initialneighborhood word hits act as starting points to find longer HSPscontaining them. The word hits are expanded in both directions alongeach of the two sequences being compared for as far as the cumulativealignment score can be increased. Extension of the word hits is stoppedwhen: the cumulative alignment score falls off by the quantity X from amaximum achieved value; the cumulative score goes to zero or below; orthe end of either sequence is reached. The BLAST algorithm parameters W,T, and X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a wordlength (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and acomparison of both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90.5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid sequence is considered similar to a phytasenucleic acid sequence of this invention if the smallest sum probabilityin a comparison of the test nucleic acid sequence to a phytase nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001. Where the test nucleicacid sequence encodes a phytase polypeptide, it is considered similar toa specified phytase nucleic acid sequence if the comparison results in asmallest sum probability of less than about 0.5, and more preferablyless than about 0.2.

The phrase “substantially identical” in the context of two nucleic acidsequences or polypeptides thus typically means that a polynucleotide orpolypeptide comprises a sequence that has at least 60% sequenceidentity, preferably at least 80%, more preferably at least 90% and mostpreferably at least 95%, compared to a reference sequence using theprograms described above (e.g., BLAST, ALIGN, CLUSTAL) using standardparameters. One indication that two polypeptides are substantiallyidentical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative substitution. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

“Hybridization” includes any process by which a strand of a nucleic acidsequence joins with a second nucleic acid sequence strand throughbase-pairing. Thus, strictly speaking, the term refers to the ability ofa target sequence to bind to a test sequence, or vice-versa.

“Hybridization conditions” are typically classified by degree of“stringency” of the conditions under which hybridization is measured.The degree of stringency can be based, for example, on the calculated(estimated) melting temperature (Tm) of the nucleic acid sequencebinding complex or probe. Calculation of Tm is well known in the art(see, e.g. page 9.50-9.51 of Sambrook (1989), below). For example,“maximum stringency” typically occurs at about Tm−5° C. (50 below the Tmof the probe); “high stringency” at about 5-10° below the Tm;“intermediate stringency” at about 10-20° below the Tm of the probe; and“low stringency” at about 20-25° below the Tm. In general, hybridizationconditions are carried out under high ionic strength conditions, forexample, using 6×SSC or 6×SSPE. Under high stringency conditions,hybridization is followed by two washes with low salt solution, forexample 0.5×SSC, at the calculated temperature. Under medium stringencyconditions, hybridization is followed by two washes with medium saltsolution, for example 2×SSC. Under low stringency conditions,hybridization is followed by two washes with high salt solution, forexample 6×SSC. Functionally, maximum stringency conditions may be usedto identify nucleic acid sequences having strict identity or near-strictidentity with the hybridization probe; while high stringency conditionsare used to identify nucleic acid sequences having about 80% or moresequence identity with the probe.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively high temperature conditions. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Press (1989); Sambrook et al.,Molecular Cloning, A Laboratory Manual, 3d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001) incorporated herein byreference.

The term “complementary”, in the context of a nucleic acid sequence,means a nucleic acid sequence having a sequence relationship to a secondnucleic acid sequence such that there is perfect alignment ofWatson-Crick base pairs along the entire length of both nucleic acidsequences.

The term “isolated” or “purified” means that a material is removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, the material is said to be “purified”when it is present in a particular composition in a higher or lowerconcentration than exists in a naturally occurring or wild type organismor in combination with components not normally present upon expressionfrom a naturally occurring or wild type organism. For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Such polynucleotides could be part of a vector,and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. A nucleic acid sequence or proteinis said to be purified, for example, if it gives rise to essentially oneband in an electrophoretic gel.

The present invention provides for the production of recombinant nucleicacids and proteins. By “recombinant” and grammatical equivalents thereofis meant produced using recombinant technology, whereby novel nucleicacids are made (recombinant nucleic acids) and proteins are producedtherefrom (recombinant proteins). Such techniques are well known in theart and many are described in great detail herein. In a broad sense, arecombinant nucleic acid sequence may be any nucleic acid sequence notin its naturally occurring form, whether it be a sequence isolated fromits naturally occurring adjoining sequence, or combined with othersequences with which it was not joined in nature to form a new nucleicacid sequence, such as in a vector. Recombinant nucleic acid sequencesalso include those that are produced from recombinant nucleic acidsequences, for example complementary sequences made throughpolymerization, additional copies made though replication, or RNAtranscribed from recombinant DNA. Recombinant protein is proteinproduced by translation of recombinant nucleic acid sequences.

As used herein in referring to phytate hydrolyzing enzymes (phytases),the term “derived from” is intended not only to indicate a phytaseproduced or producible by a strain of the organism in question, but alsoa phytase encoded by a DNA sequence isolated from such strain andproduced in a host organism containing such DNA sequence. Additionally,the term is intended to indicate a phytase which is encoded by a DNAsequence of synthetic and/or cDNA origin and which has the identifyingcharacteristics of the phytase in question. To exemplify, “phytasesderived from Penicillium” refers to those enzymes having phytaseactivity which are naturally-produced by Penicillium, as well as tophytases like those produced by Penicillium sources but which throughthe use of genetic engineering techniques are produced bynon-Penicillium organisms transformed with a nucleic acid sequenceencoding said phytases. The present invention encompasses phytatehydrolyzing enzymes that are equivalent to those that are derived fromthe particular microbial strain mentioned. Being “equivalent,” in thiscontext, means that the phytate hydrolyzing enzymes are encoded by apolynucleotide capable of hybridizing to the polynucleotide having thesequence as shown in any one of FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7,and 9, respectively) under conditions of medium to high stringency.Being equivalent means that the phytate hydrolyzing enzyme comprises atleast 55% identity, preferably at least 60% identity, more preferably atleast 65% identity, still more preferably at least 70% identity, yetmore preferably at least 75% identity, even more preferably at least 80%identity, again more preferably at least 85% identity, yet again morepreferably at least 90% identity, and most preferably at least 95% up toabout 100% identity to the phytate hydrolyzing enzyme having the aminoacid sequence disclosed in one of FIGS. 2, 3, 5, 6, 8, 10 and 11 (SEQ IDNO:2, 3, 5, 6, 8, 10, and 11, respectively). The present invention alsoencompasses mutants, variants and derivatives of the phytate hydrolyzingenzymes of the present invention as long as the mutant, variant orderivative phytate hydrolyzing enzyme is able to retain at least onecharacteristic activity of the naturally occurring phytate hydrolyzingenzyme. As used herein, the term “mutants and variants”, when referringto phytate hydrolyzing enzymes, refers to phytate hydrolyzing enzymesobtained by alteration of the naturally occurring amino acid sequenceand/or structure thereof, such as by alteration of the DNA nucleotidesequence of the structural gene and/or by direct substitution and/oralteration of the amino acid sequence and/or structure of the phytatehydrolyzing enzyme.

The term “derivative” or “functional derivative” as it relates tophytase is used herein to indicate a derivative of phytase which has thefunctional characteristics of phytase of the present invention.Functional derivatives of phytase encompass naturally occurring,synthetically or recombinantly produced peptides or peptide fragments,mutants or variants which may have one or more amino acid deletions,substitutions or insertions which have the general characteristics ofthe phytase of the present invention.

The term “functional derivative” as it relates to nucleic acid sequencesencoding phytase is used throughout the specification to indicate aderivative of a nucleic acid sequence which has the functionalcharacteristics of a nucleic acid sequence which encodes phytase.Functional derivatives of a nucleic acid sequence which encode phytaseof the present invention encompass naturally occurring, synthetically orrecombinantly produced nucleic acid sequences or fragments, mutants orvariants thereof which may have one or more nucleic acid deletions,substitutions or insertions and encode phytase characteristic of thepresent invention. Variants of nucleic acid sequences encoding phytaseaccording to the invention include alleles and variants based on thedegeneracy of the genetic code known in the art. Mutants of nucleic acidsequences encoding phytase according to the invention include mutantsproduced via site-directed mutagenesis techniques (see for example,Botstein, D. and Shortle, D., 1985, Science 229:1193-1201 and Myers, R.M., Lerman, L. S., and Maniatis, T., 1985, Science 229: 242-247),error-prone PCR (see for example, Leung, D. W., Chen, E., and Goeddel,D. V., 1989, Technique 1: 11-15; Eckert, K. A. and Kunkel, T. A., 1991,PCR Methods Applic. 1: 17-24; and Cadwell, R. C. and Joyce, G. F., 1992,PCR Methods Applic. 2: 28-33) and/or chemical-induced mutagenesistechniques known in the art (see for example, Elander, R. P., Microbialscreening, Selection and Strain Improvement, in Basic Biotechnology, J.Bullock and B. Kristiansen Eds., Academic Press, New York, 1987, 217).

“Expression vector” means a DNA construct comprising a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of the DNA in a suitable host. Such controlsequences may include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable ribosome-binding sites on the mRNA, and sequences which controltermination of transcription and translation. Different cell types arepreferably used with different expression vectors. A preferred promoterfor vectors used in Bacillus subtilis is the AprE promoter; a preferredpromoter used in E. coli is the Lac promoter and a preferred promoterused in Aspergillus niger is glaA. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may, under suitable conditions, integrate into thegenome itself.

In the present specification, plasmid and vector are sometimes usedinterchangeably. However, the invention is intended to include otherforms of expression vectors which serve equivalent functions and whichare, or become, known in the art. Thus, a wide variety ofhost/expression vector combinations may be employed in expressing theDNA sequences of this invention. Useful expression vectors, for example,may consist of segments of chromosomal, non-chromosomal and syntheticDNA sequences such as various known derivatives of SV40 and knownbacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1,pBR322, pMb9, pUC19 and their derivatives, wider host range plasmids,e.g., RP4, phage DNAs e.g., the numerous derivatives of phage 1, e.g.,NM989, and other DNA phages, e.g., M13 and filamentous single strandedDNA phages, yeast plasmids such as the 2 m plasmid or derivativesthereof, vectors useful in eukaryotic cells, such as vectors useful inanimal cells and vectors derived from combinations of plasmids and phageDNAs, such as plasmids which have been modified to employ phage DNA orother expression control sequences.

Expression techniques using the expression vectors of the presentinvention are known in the art and are described generally in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Press (1989); Sambrook et al., MolecularCloning, A Laboratory Manual, 3d Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001). Often, such expression vectorsincluding the DNA sequences of the invention are transformed into aunicellular host by direct insertion into the genome of a particularspecies through an integration event (see e.g., Bennett & Lasure, MoreGene Manipulations in Fungi, Academic Press, San Diego, pp. 70-76 (1991)and articles cited therein describing targeted genomic insertion infungal hosts, incorporated herein by reference).

“Host strain” or “host cell” means a suitable host for an expressionvector comprising DNA according to the present invention. Host cellsuseful in the present invention are generally procaryotic or eucaryotichosts, including any transformable microorganism in which expression canbe achieved. For example, host strains can be Bacillus subtilis,Escherichia coli, Trichoderma iongibrachiatum, Saccharomyces cerevisiae,Aspergillus niger, and Aspergillus nidulans. Host cells are transformedor transfected with vectors constructed using recombinant DNAtechniques. Such transformed host cells are capable of both replicatingvectors encoding phytase and its variants (mutants) or expressing thedesired peptide product.

Examples of appropriate expression hosts include: bacterial cells, suchas E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such asAspergillus and Penicillium; insect cells such as Drosophila andSpodoptera Sf9; animal cells such as CHO, COS, HEK 293 or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein. It should be noted that the invention is not limitedby the particular host cells employed.

II. Phytase Enzymes and Nucleic Acid Sequences Encoding Phytase Enzymes

One aspect of the present invention provides proteins or polypeptideswhich are capable of catalyzing the hydrolysis of phytate and releasinginorganic phosphate; for example, enzymes having catalytic activity asdefined in Enzyme Commission EC number 3.1.3.8, or in EC number3.1.3.26. In one preferred embodiment, the invention provides aso-called 3-phytase. The present invention additionally encompassespolynucleotides (e.g., DNA) which encode such phytate hydrolyzingproteins or polypeptides.

Preferably, the phytase and/or polynucleotides encoding the phytaseaccording to the present invention is derived from a fungus, preferablyfrom an anaerobic fungus or thermophilic fungus and most preferably fromPenicillium sp., e.g., Penicillium chrysogenum, Fusarium sp., e.g.,Fusarium javanicum or Fusarium vertisillibodes or Humicola sp., e.g.Humicola grisea. Thus, it is contemplated that the phytase or the DNAencoding the phytase according to the invention can be derived fromAbsidia sp.; Acremonium sp.; Actinomycetes sp.; Agaricus sp.;Anaeromyces sp.; Aspergillus sp., including A. auculeatus, A. awamori,A. flavus, A. foetidus, A. fumaricus, A. fumigatus, A. nidulans, A.niger, A. oryzae, A. terreus and A. versicolor; Aeurobasidium sp.;Cephalosporum sp.; Chaetomium sp.; Coprinus sp.; Dactyllum sp.; Fusariumsp., including F. conglomerans, F. decemcellulare, F. javanicum, F.lini, F. oxysporum and F. solani; Gliocladium sp.; Humicola sp.,including H. insolens and H. lanuginosa; Mucor sp.; Myceliopthora ssp.,including M. thermophila; Neurospora sp., including N. crassa and N.sitophila; Neocallimastix sp.; Orpinomyces sp.; Penicillium spp;Phanerochaete sp.; Phlebia sp.; Piromyces sp.; Pseudomonas sp.; Rhizopussp.; Schizophyllum sp.; Streptomyces spp; Trametes sp.; and Trichbdermasp., including T. reesei, T. longibrachiatum and T. viride; andZygorhynchus sp. Similarly, it is envisioned that a phytase and/or DNAencoding a phytase as described herein may be derived from bacteria suchas Streptomyces sp., including S. olivochromogenes; specifically fiberdegrading ruminal bacteria such as Fibrobacter succinogenes; and inyeast including Candida torresii; C. parapsllosis; C. sake; C.zeylanoides; Pichia minuta; Rhodotorula glutinis; R. mucilaginosa; andSporobolomyces holsaticus.

In one preferred embodiment, the phytase and/or polynucleotides encodingthe phytase according to the present invention is/are derived from (i) agrain-spoilage fungus, such as Penicillium hordei, Penicillium piceum,or Penicillium brevi-compactum; or (ii) an ectomycorrhizal fungusassociated with tree roots, e.g., Laccaria laccata, Laccaria rufus,Paxillus involutus, Hebeloma crustuliniforme, Amanita rubescens, orAmanita muscaria. According to a preferred embodiment, the phytaseand/or polynucleotide encoding the phytase of the present invention isin a purified form, i.e., present in a particular composition in ahigher or lower concentration than exists in a naturally occurring orwild type organism or in combination with components not normallypresent upon expression from a naturally occurring or wild typeorganism.

The invention encompasses phytate hydrolyzing proteins and peptidescomprising at least 55% identity, preferably at least 60% identity, morepreferably at least 65% identity, still more preferably at least 70%identity, yet more preferably at least 75% identity, even morepreferably at least 80% identity, again more preferably at least 85%identity, yet again more preferably at least 90% identity, and mostpreferably at least 95% up to about 100% identity to the phytatehydrolyzing enzyme having the amino acid sequence disclosed in FIGS. 2,3, 5, 6, 8, 10 or 11 (SEQ ID NO:2, 3, 5, 6, 8, 10, and 11,respectively).

The invention further encompasses polynucleotides, e.g., DNA, whichencode phytate hydrolyzing enzymes derived from fungal sources, such asPenicillium sp., which polynucleotides include a sequence having atleast 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity and atleast 9.5% identity to the polynucleotide sequence disclosed in any oneof FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7, and 9, respectively), as longas the enzyme encoded by the polynucleotide is capable of catalyzing thehydrolysis of phytate and releasing inorganic phosphate. In a preferredembodiment, the polynucleotide encoding the phytate hydrolyzing enzymehas the polynucleotide sequence as shown in any one of FIGS. 1, 4, 7 and9 (SEQ ID NO:1, 4, 7, and 9, respectively), or is capable of hybridizingto the polynucleotide sequence as shown in any one of FIGS. 1, 4, 7 and9 (SEQ ID NO:1, 4, 7, and 9, respectively) or its complement, or iscomplementary to the polynucleotide sequence as shown in any one ofFIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7, and 9, respectively). As will beunderstood by the skilled artisan, due to the degeneracy of the geneticcode, a variety of polynucleotides can encode the phytate hydrolyzingenzyme disclosed in any one of FIGS. 2, 3, 5, 6, 8, 10 and 11 (SEQ IDNO:2, 3, 5, 6, 8, 10, and 11, respectively). The present inventionencompasses all such polynucleotides.

III. Obtaining Polynucleotides Encoding a Phytate Hydrolyzing Enzyme

The nucleic acid sequence encoding a phytate hydrolyzing enzyme may beobtained by standard procedures known in the art from, for example,cloned DNA (e.g., a DNA “library”), by chemical synthesis, by cDNAcloning, by PCR, or by the cloning of genomic DNA, or fragments thereof,purified from a desired cell, such as a fungal species (See, forexample, Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual,3d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Glover, D M and Hames, B D (Eds.), 1995, DNA Cloning 1: A PracticalApproach and DNA Cloning 2: A Practical Approach, Oxford UniversityPress, Oxford). Nucleic acid sequences derived from genomic DNA maycontain regulatory regions in addition to coding regions.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will comprise at least a portion of the desiredgene. The DNA may be cleaved at specific sites using various restrictionenzymes. Alternatively, one may use DNAse in the presence of manganeseto fragment the DNA, or the DNA can be physically sheared, as forexample, by sonication. The linear DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis, PCR and columnchromatography.

Once nucleic acid sequence fragments are generated, identification ofthe specific DNA fragment encoding a phytate hydrolyzing enzyme may beaccomplished in a number of ways. For example, a phytate hydrolyzingenzyme encoding gene of the present invention or its specific RNA, or afragment thereof, such as a probe or primer, may be isolated and labeledand then used in hybridization assays to detect a generated gene.(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. andHogness, D., 1975, Proc. Natl. Acad. Sci. USA 72:3961). Those DNAfragments sharing substantial sequence similarity to the probe willhybridize under medium to high stringency.

The present invention encompasses phytate hydrolyzing enzymes derivedfrom fungal species (esp., Penicillium, Fusarium and Humicola species)which are identified through nucleic acid sequence hybridizationtechniques using one of the sequences disclosed in FIGS. 1, 4, 7 and 9(SEQ ID NO:1, 4, 7, and 9, respectively), or a suitable portion orfragment thereof (e.g., at least about 10-15 contiguous nucleotides), asa probe or primer and screening nucleic acid sequences of either genomicor cDNA origin. Nucleic acid sequences encoding phytate hydrolyzingenzymes derived from fungal species and having at least 65% identity tothe sequence of one of FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7, and 9,respectively) or a portion or fragment thereof can be detected byDNA-DNA or DNA-RNA hybridization or amplification using probes, portionsor fragments of the disclosed sequences. Accordingly, the presentinvention provides a method for the detection of nucleic acid sequencesencoding a phytate hydrolyzing enzyme encompassed by the presentinvention which comprises hybridizing part or all of a nucleic acidsequence of FIG. 1, 4, 7 or 9 (SEQ ID NO:1, 4, 7, and 9, respectively)with a nucleic acid sequence of either genomic or cDNA origin.

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence disclosed in FIGS. 1, 4, 7 or 9 (SEQ ID NO:1, 4, 7,and 9, respectively) under conditions of medium to high stringency. Inone embodiment, hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid sequence binding complex, as taughtin Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.)incorporated herein by reference, and confer a defined stringency. Inthis embodiment, “maximum stringency” typically occurs at about Tm−5° C.(5° C. below the Tm of the probe); “high stringency” at about 5° C. to10° C. below Tm; “medium” or “intermediate stringency” at about 10° C.to 20° C. below Tm; and “low stringency” at about 20° C. to 25° C. belowTm. A maximum stringency hybridization can be used to identify or detectidentical or near-identical polynucleotide sequences, while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach C W and G S Dveksler(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y.). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from the sequences ofFIG. 1, 4, 7 or 9 (SEQ ID NO:1, 4, 7, and 9, respectively), preferablyabout 12 to 30 nucleotides, and more preferably about 25 nucleotides canbe used as a probe or PCR primer.

A preferred method of isolating a nucleic acid sequence construct of theinvention from a cDNA or genomic library is by use of polymerase chainreaction (PCR) using degenerate oligonucleotide probes prepared on thebasis of the amino acid sequence of the protein having the amino acidsequence shown in any one of FIGS. 2, 3, 5, 6, 8, 10 and 11 (SEQ IDNO:2, 3, 5, 6, 8, 10, and 11, respectively). For instance, the PCR maybe carried out using the techniques described in U.S. Pat. No.4,683,202.

In view of the above, it will be appreciated that the polynucleotidesequences provided in FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7, and 9,respectively) are useful for obtaining identical or homologous fragmentsof polynucleotides from other species, and particularly from fungi(e.g., the grain-spoilage fungi, or the Ectomycorrhizae) which encodeenzymes having phytase activity.

IV. Obtaining Derivative or Variant Phytate Hydrolyzing Enzymes

In one embodiment, the phytase proteins are derivative or variantphytase as compared to the wild-type sequence. That is, as outlined morefully below, the derivative phytase peptide will contain at least oneamino acid substitution, deletion or insertion, with amino acidsubstitutions being particularly preferred. The amino acid substitution,insertion or deletion may occur at any residue within the phytasepeptide.

Also included in an embodiment of phytase proteins of the presentinvention are amino acid sequence variants. These variants fall into oneor more of three classes: substitutional, insertional or deletionalvariants. These variants ordinarily are prepared by site specificmutagenesis of nucleotides in the DNA encoding the phytase protein,using cassette or PCR mutagenesis or other techniques well known in theart, to produce DNA encoding the variant, and thereafter expressing theDNA in recombinant cell culture as outlined above. However, variantphytase protein fragments having up to about 100-150 residues may beprepared by in vitro synthesis using established techniques. Amino acidsequence variants are characterized by the predetermined nature of thevariation, a feature that sets them apart from naturally occurringallelic or interspecies variation of the phytase protein amino acidsequence. The variants typically exhibit the same qualitative biologicalactivity as the naturally occurring analogue, although variants can alsobe selected which have modified characteristics as will be more fullyoutlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of phytaseprotein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated, and may occurinternally or at either terminus of the encoded protein. Deletions rangefrom about 1 to about 20 residues, although in some cases deletions maybe much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the phytase are desired,substitutions are generally made in accordance with the following chartof conservative substitution residues: CHART I Original ResidueExemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser GlnAsn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln,Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, PheVal Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand may elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the phytase proteins as needed. Alternatively, thevariant may be designed such that the biological activity of the phytaseis altered. For example, glycosylation sites may be altered or removed.Such alterations may result in altered immunogenicity, as well.

Covalent modifications of phytase polypeptides are included within thescope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a phytase polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of a phytase polypeptide.Derivatization with bifunctional agents is useful, for instance, forcrosslinking a phytase to another protein. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl, threonyl or tyrosyl residues, methylation ofthe α-amino groups of lysine, arginine, and histidine side chains [T.E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the phytase polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native phytase, and/or addingone or more glycosylation sites that are not present in the nativepolypeptide.

Addition of glycosylation sites to polypeptides may be accomplished byaltering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence phytase polypeptide (forO-linked glycosylation sites). The phytase amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the phytase polypeptide at preselected basessuch that codons are generated that will translate into the desiredamino acids.

Another means of increasing the number of carbohydrate moieties on thephytase polypeptide is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, Crit. Rev.Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the phytase may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of phytase comprises linking thephytase polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Phytases of the present invention may also be modified to form chimericmolecules comprising a phytase polypeptide fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of a phytase polypeptide with atag polypeptide which provides an epitope to which an anti-tag antibodycan selectively bind. The epitope tag is generally placed at theamino-or carboxyl-terminus of the phytase polypeptide. The presence ofsuch epitope-tagged forms of a phytase can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe phytase to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In preferred embodiment, the chimeric molecule may comprisea fusion of a phytase polypeptide with an initial sequence or signalpolypeptide, such as a secretion signal, of a different phytase or otherprotein. The fusion may involve the addition of a sequence from aprotein, such as a phytase, which is native to the host cell in whichthe phytase is being expressed. Specific examples of this are providedin the Examples section, below.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

Also included with the definition of phytase in one embodiment are otherphytase proteins from other organisms, which are cloned and expressed asoutlined below. Thus, probe or degenerate polymerase chain reaction(PCR) primer sequences may be used to find other related phytases fromfungi or other organisms. As will be appreciated by those in the art,particularly useful probe and/or PCR primer sequences include the highlyconserved amino acid sequences and the known binding or catalyticsequences. For example, the phosphate binding region of phytase producedin various fungi is highly conserved. As is generally known in the art,preferred PCR primers are from about 15 to about 35 nucleotides inlength, with from about 20 to about 30 being preferred, and may containinosine as needed. The conditions for the PCR reaction are well known inthe art.

V. Expression and Recovery of Phytate Hydrolyzing-Enzymes

The polynucleotide sequences of the present invention may be expressedby operatively linking them to an expression control sequence in anappropriate expression vector and employed in that expression vector totransform an appropriate host according to techniques well establishedin the art. The polypeptides produced on expression of the DNA sequencesof this invention can be isolated from the fermentation of cell culturesand purified in a variety of ways according to well establishedtechniques in the art. One of skill in the art is capable of selectingthe most appropriate isolation and purification techniques.

More particularly, the present invention provides host cells, expressionmethods and systems for the production of phytate hydrolyzing enzymesderived from microorganisms, such as Penicillium, Fusarium and Humicolaspecies. Once a nucleic acid sequence encoding a phytate hydrolyzingenzyme of the present invention is obtained, recombinant host cellscontaining the nucleic acid sequence may be constructed using techniqueswell known in the art. Molecular biology techniques are disclosed inSambrook et al., Molecular Biology Cloning: A Laboratory Manual, SecondEdition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Sambrook et al., Molecular Cloning, A Laboratory Manual,3d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

In one embodiment, nucleic acid sequences encoding phytate hydrolyzingenzymes derived from Penicillium, Fusarium and Humicola species andhaving at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90% and at least 95% identity to the nucleic acid sequenceof any one of FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7, and 9,respectively) or a functional derivative thereof, or which is capable ofhybridizing under conditions of intermediate to high stringency to thenucleic acid sequence of any one of FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4,7, and 9, respectively), or which is complementary to the nucleic acidsequence of any one of FIGS. 1, 4, 7 and 9 (SEQ ID NO:1, 4, 7, and 9,respectively) is obtained and transformed into a host cell usingappropriate vectors.

The nucleic acid sequences encoding phytate hydrolyzing enzymes caninclude a leader sequence capable of providing for the secretion of theencoded phytase. Depending on whether the phytase is to be expressedintracellularly or is secreted, a DNA sequence or expression vector ofthe invention can be engineered such that the mature form of the phytaseis expressed with or without a natural phytase signal sequence or asignal sequence which functions in a fungus (e.g., Aspergillus niger,other prokaryotes or eukaryotes. Expression can also be achieved byeither removing or partially removing said signal sequence.

A variety of vectors and transformation and expression cassettessuitable for the cloning, transformation and expression in fungus,yeast, bacteria, insect and plant cells are known by those of skill inthe art. Typically, the vector or cassette contains sequences directingtranscription and translation of the nucleic acid sequence, a selectablemarker, and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. These controlregions may be derived from genes homologous or heterologous to the hostas long as the control region selected is able to function in the hostcell.

Initiation control regions or promoters, which are useful to driveexpression of the phytate hydrolyzing enzymes in a host cell are knownto those skilled in the art. A nucleic acid sequence encoding thephytate hydrolyzing enzyme is linked operably through initiation codonsto selected expression control regions for effective expression of suchenzyme. Once suitable cassettes are constructed, they are used totransform the host cell.

In cases where plant expression vectors are used, the expression of asequence encoding phytase may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMV(Brisson et al. (1984) Nature 310:511-514) may be used alone or incombination with the omega leader sequence from TMV (Takamatsu et al.(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680;Broglie et al. (1984) Science 224:838-843); or heat shock promoters(Winter J and Sinibaldi R M (1991) Results Probl Cell Differ 17:85-105)may be used. These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. For reviewsof such techniques, see Hobbs S or Murry L E (1992) in McGraw HillYearbook of Science and Technology, McGraw Hill, New York, N.Y., pp191-196; or Weissbach and Weissbach (1988) Methods for Plant MolecularBiology, Academic Press, New York, N.Y., pp 421-463.

General transformation procedures are taught in Current Protocols InMolecular Biology (3rd edition, edited by Ausubel et al., John Wiley &Sons, Inc. 1995, Chapter 9) and include calcium phosphate methods,transformation using PEG and electroporation.

For Aspergillus and Trichoderma, PEG and Calcium mediated protoplasttransformation can be used (Finkelstein, D B 1992 Transformation. InBiotechnology of Filamentous Fungi. Technology and Products (eds byFinkelstein & Bill) 113-156. Electroporation of protoplast is disclosedin Finkelestein, D B 1992 Transformation. In Biotechnology ofFilamentous Fungi. Technology and Products (eds by Finkelstein & Bill)113-156. Microprojection bombardment on conidia is described in Fungaroet al. (1995) Transformation of Aspergillus nidulans by microprojectionbombardment on intact conidia, FEMS Microbiology Letters 125 293-298.Agrobacterium mediated transformation is disclosed in Groot et al.(1998) Agrobacterium tumefaciens-mediated transformation of filamentousfungi, Nature Biotechnology 16 839-842 and U.S. Pat. No. 6,255,115. Fortransformation of Saccharomyces, lithium acetate mediated transformationand PEG and calcium mediated protoplast transformation as well aselectroporation techniques are known by those of skill in the art.

Host cells which contain the coding sequence for a phytate hydrolyzingenzyme of the present invention and express the protein may beidentified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridization and protein bioassay or immunoassay techniqueswhich include membrane-based, solution-based, or chip-based technologiesfor the detection and/or quantification of the nucleic acid sequence orprotein.

It should also be noted that the invention contemplates in vitroexpression of the phytase enzymes described herein.

In preferred embodiments of the invention, phytase is produced in fungalcells. In one embodiment of the present invention, a polynucleotidesequence encoding a phytate hydrolyzing enzyme derived from Penicilliumchrysogenum (deposit No. NRRL 1951) is isolated and expressed inAspergillus niger, and in another embodiment is expressed in Aspergillusnidulans. In another embodiment, a polynucleotide sequence encoding aphytate hydrolyzing enzyme derived from Fusarium javanicum (deposit No.CBS 203.32) or Fusarium vertisillibodes is isolated and expressed. Inyet another embodiment, a polynucleotide sequence encoding a phytatehydrolyzing enzyme derived from Humicola grisea (deposit No. ATCC 22081)is isolated and expressed. The expressed phytase can then be recovered,e.g., as described below.

In preferred embodiments of the invention, the phytase is expressed inplants. Transgenic plant, as used herein, refers to a plant thatcontains recombinant genetic material not normally found in plants ofthis type and which has been introduced into the plant in question (orinto progenitors of the plant) by human manipulation. Thus, a plant thatis grown from a plant cell into which recombinant DNA is introduced bytransformation is a transgenic plant, as are all offspring of that plantthat contain the introduced transgene (whether produced sexually orasexually). It is understood that the term transgenic plant encompassesthe entire plant and parts of said plant, for instance grains, seeds,flowers, leaves, roots, fruit, pollen, stems, etc.

The present invention is applicable to both dicotyledonous plants (e.g.tomato, potato, soybean, cotton, tobacco, etc.) and monocotyledonousplants, including, but not limited to graminaceous monocots such aswheat (Triticum spp.), rice (Oryza spp.), barley (Hordeum spp.), oat(Avena spp.), rye (Secale spp.), corn (Zea mays), sorghum (Sorghum spp.)and millet (Pennisetum spp). For example, the present invention can beemployed with barley genotypes including, but not limited to Morex,Harrington, Crystal, Stander, Moravian III, Galena, Salome, Steptoe,Klages, Baronesse, and with wheat genotypes including, but not limitedto Yecora Rojo, Bobwhite, Karl and Anza. In general, the invention isparticularly useful in cereals.

Standard molecular biology methods and plant transformation techniquescan be used to produce transgenic plants that produce seeds containingphytase protein. The following description provides general guidance asto the selection of particular constructs and transformation procedures.

The present invention utilizes recombinant constructs that are suitablefor obtaining expression of phytase in plant seeds relative tonon-transformed plant seeds. In their most basic form, these constructsmay be represented as Pr-Ph, wherein Pr is a seed-specific promoter andPh is a nucleic acid sequence encoding phytase. In another embodiment, apeptide signal sequence that targets expression of the phytasepolypeptide to an intracellular body may be employed. Such constructsmay be represented as Pr-SS-Ph, wherein SS is the signal peptide.Nucleic acid molecules that may be used as the source of each of thesecomponents are described in the Definitions section above.

Each component is operably linked to the next. For example, where theconstruct comprises the hordein D-promoter (P), the hordein D-signalsequence (SS) encoding the hordein signal peptide, and an open readingframe encoding a phytase (Ph), the hordein promoter is linked to the 5′end of the sequence encoding the hordein signal sequence, and thehordein signal sequence is operably linked to the 5′ end of the phytaseopen reading frame, such that C terminus of the signal peptide is joinedto the N-terminus of the encoded protein.

The construct will also typically include a transcriptional terminationregion following the 3′ end of the encoded protein ORF. Illustrativetranscriptional termination regions include the nos terminator fromAgrobacterium Ti plasmid and the rice alpha-amylase terminator.

Standard molecular biology methods, such as the polymerase chainreaction, restriction enzyme digestion, and/or ligation may be employedto produce these constructs comprising any nucleic acid molecule orsequence encoding a phytase protein or polypeptide.

Introduction of the selected construct into plants is typically achievedusing standard transformation techniques. The basic approach is to: (a)clone the construct into a transformation vector; which (b) is thenintroduced into plant cells by one of a number of techniques (e.g.,electroporation, microparticle bombardment, Agrobacterium infection);(c) identify the transformed plant cells; (d) regenerate whole plantsfrom the identified plant cells, and (d) select progeny plantscontaining the introduced construct. Preferably all or part of thetransformation vector will stably integrate into the genome of the plantcell. That part of the transformation vector which integrates into theplant cell and which contains the introduced Pr-Ph or Pr-SS-Ph sequence(the introduced “phytase transgene”) may be referred to as therecombinant expression cassette.

Selection of progeny plants containing the introduced transgene may bemade based upon the detection of phytase expression in seeds, or uponenhanced resistance to a chemical agent (such as an antibiotic) as aresult of the inclusion of a dominant selectable marker geneincorporated into the transformation vector.

Successful examples of the modification of plant characteristics bytransformation with cloned nucleic acid sequences are replete in thetechnical and scientific literature. Selected examples, which serve toillustrate the knowledge in this field of technology include:

-   -   U.S. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and        Methods”);    -   U.S. Pat. No. 5,677,175 (“Plant Pathogen Induced Proteins”);    -   U.S. Pat. No. 5,510,471 (“Chimeric Gene for the Transformation        of Plants”);    -   U.S. Pat. No. 5,750,386 (“Pathogen-Resistant Transgenic        Plants”);    -   U.S. Pat. No. 5,597,945 (“Plants Genetically Enhanced for        Disease Resistance”);    -   U.S. Pat. No. 5,589,615 (“Process for the Production of        Transgenic Plants with Increased Nutritional Value Via the        Expression of Modified 2S Storage Albumins”);    -   U.S. Pat. No. 5,750,871 (“Transformation and Foreign Gene        Expression in Brassica Species”);.    -   U.S. Pat. No. 5,268,526 (“Over expression of Phytochrome in        Transgenic Plants”);    -   U.S. Pat. No. 5,780,708 (“Fertile Transgenic Com Plants”);    -   U.S. Pat. No. 5,538,880 (“Method For Preparing Fertile        Transgenic Corn Plants”);    -   U.S. Pat. No. 5,773,269 (“Fertile Transgenic Oat Plants”);    -   U.S. Pat. No. 5,736,369 (“Method For Producing Transgenic Cereal        Plants”);    -   U.S. Pat. No. 5,610,049 (“Methods For Stable Transformation of        Wheat”).

These examples include descriptions of transformation vector selection,transformation techniques and the construction of constructs designed toexpress an introduced transgene.

The transgene-expressing constructs of the present invention may beusefully expressed in a wide range of higher plants to obtain seed- orgrain-specific expression of selected polypeptides. The invention isexpected to be particularly applicable to monocotyledonous cereal plantsincluding barley, wheat, rice, rye, maize, triticale, millet, sorghum,oat, forage, and turf grasses. In particular, the transformation methodsdescribed herein will enable the invention to be used with genotypes ofbarley including Morex, Harrington, Crystal, Stander, Moravian III,Galena, Golden Promise, Steptoe, Klages and Baronesse, and commerciallyimportant wheat genotypes including Yecora Rojo, Bobwhite, Karl andAnza.

The invention may also be applied to dicotyledenous plants, including,but not limited to, soybean, sugar beet, cotton, beans, rape/canola,alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut,clover; vegetables such as lettuce, tomato, cucurbits, cassaya, potato,carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brusselssprouts, peppers; and tree fruits such as citrus, apples, pears,peaches, apricots, and walnuts.

A number of recombinant vectors suitable for stable transformation ofplant cells or for the establishment of transgenic plants have beendescribed including those described in Weissbach and Weissbach, (1988),and Gelvin et al., J. Bacteriol. 172(3):1600-1608 (1990). Typically,plant transformation vectors include one or more ORFs under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker with 5′ and 3′ regulatory sequences. The selection ofsuitable 5′ and 3′ regulatory sequences for constructs of the presentinvention is discussed above. Dominant selectable marker genes thatallow for the ready selection of transformants include those encodingantibiotic resistance genes (e.g., resistance to hygromycin, kanamycin,bleomycin, G418, streptomycin or spectinomycin) and herbicide resistancegenes (e.g, phosphinothricin acetyltransferase).

Methods for the transformation and regeneration of both monocotyledonousand dicotyledonous plant cells are known, and the appropriatetransformation technique will be determined by the practitioner. Thechoice of method will vary with the type of plant to be transformed;those skilled in the art will recognize the suitability of particularmethods for given plant types. Suitable methods may include, but are notlimited to: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG) mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium mediated transformation. Typical procedures fortransforming and regenerating plants are described in the patentdocuments listed at the beginning of this section.

Following transformation, transformants are preferably selected using adominant selectable marker. Typically, such a marker will conferantibiotic or herbicide resistance on the seedlings of transformedplants, and selection of transformants can be accomplished by exposingthe seedlings to appropriate concentrations of the antibiotic orherbicide. After transformed plants are selected and grown to maturityto allow seed set, the seeds can be harvested and assayed for expressionof phytase.

The phytase of the invention can be recovered from culture medium orfrom host cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or byenzymatic cleavage. Cells employed in expression of phytase can bedisrupted by various physical or chemical means, such as freeze-thawcycling, sonication, mechanical disruption, or cell lysing agents. Itmay be desired to purify the phytase from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants; andmetal chelating columns to bind epitope-tagged forms of the phytase.Various methods of protein purification may be employed and such methodsare known in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular form of phytase produced.

In a preferred embodiment, the phytase(s) is/are produced in transgenicnon-human animals. Methods of producing such transgenic animals aredescribed, for example, in U.S. Pat. No. 6,291,740. Methods for thesuccessful production of transgenic bovine (e.g., U.S. Pat. Nos.6,080,912 and 6,066,725), swine (e.g., U.S. Pat. Nos. 6,271,436 and5,942,435), goats (e.g., U.S. Pat. No. 5,907,080) and fish (e.g., U.S.Pat. No. 5,998,697) are available in the art. Furthermore,organ-specific expression, particularly expression in milk produced bythe transgenic animals, is within the skill of the ordinary artisan(e.g., e.g., U.S. Pat. Nos. 6,268,545 and 6,262,336). The disclosure ofeach of these patents is incorporated herein in its entirety.

VI. Assaying for Phytase Activity

Assays for phytase activity are well known in the art. Perhaps the mostwidely used is the classic assay for liberation of inorganic phosphatedeveloped by Fiske and SubbaRow, Journal of BiologicalChemistry66:375-392 (1925). A variation of this method is found inMitchell et al., Microbiol. 143:245-252 (1997). A preferred method isdescribed in Food Chemicals Codex, 4th Edition, Committee on FoodChemicals Codex, Institute of Medicine, National Academy Press,Washington, D.C., 1996 at pages 809-810. Each of these references areincorporated herein.

Generally, the assay involves allowing a measured weight or volume of aphytase sample to react with phytate in solution for a measured periodof time. The reaction is stopped and a color solution containingammonium molybdate (AM) is added to the reaction solution. Colorimetryis then performed using a spectrophotometer and compared to controls ofknown concentration of inorganic phosphate (P_(i)) and/or controlsproduced by reactions with enzymes having known phytase activity. A Unitof activity is determined as the amount of enzyme sample required toliberate 1 μmol P_(i) per minute from phytate under defined reactionconditions.

Enzyme reactions are frequently run at pH 5.5 and 37° C. However, pH andtemperature conditions may be varied to determine optimum reactionconditions and tolerances for a given phytase. When different reactionconditions are tested, Units of activity should still be related to asingle specific set of reaction conditions.

The reaction may be stopped and then the color solution added, or astop/color solution may be used that both arrests the enzyme activityand adds a product whose spectral absorbance is measurably affected bythe concentration of P_(i) in a predictable and calculatable manner. Asdiscussed above, the color solutions generally contain AM. Variousexamples of such solutions are available in the relevant literature. InU.S. Pat. No. 6,039,942, the reaction is stopped using trichloroactetate(TCA) and the color solution added thereafter contained ferrous sulfateand AM. In other examples wherein the reaction was first stopped withTCA, different color solution contained sulfuric acid, AM and ascorbicacid (U.S. Pat. No. 6,221,644) and sulfuric acid, AM and ferrous sulfate(U.S. Pat. No. 6,190,897). In other cases, the color and stop solutionare the same. For example, in both U.S. Pat. Nos. 6,139,902 and6,261,592, the solution contained sulfuric acid, AM and acetone, afterwhich a solution containing acetic acid was added. In a preferredembodiment, the color/stop solution contains ammonium vanadate, AM andnitric acid (see Food Chemicals Codex, above).

Wavelength-specific absorption by the final solution, containing thereaction solution and stop/color solution(s), is measured using aspectrophotometer. Many such instruments are available and their use isroutine in the art. The wavelength used for absorption measurement canvary with the components of the color solution. For example, thereferences cited above measured absorbance at 380, 415, 690, 700 or 750nm. Any of these may provide adequate indication of P_(i) concentrationin these solutions. However, the wavelength used should generally be theone described in a given protocol. The skilled artisan can easilydetermine empirically which wavelength provides optimum discriminationof differences in P_(i) concentration by comparing the linearity ofabsorption change between serially diluted control solutions of knownP_(i) concentration at different wavelengths.

VII. Applications of Phytate Hydrolyzing Enzymes

The phytase and derivatives thereof as taught herein can be used in avariety of applications where it is desirable to separate phosphorousfrom phytate. Several exemplary applications are set forth below.

For example, the invention provides for the use of cells or sporescapable of producing phytase according to the invention as a probioticor direct fed microbial product. Preferred embodiments for said uses arephytase-producing Aspergillus sp. of the invention.

In addition, the invention contemplates the use of phytase as describedherein in food or animal feed.

The present invention provides food or animal feed including phytase asdescribed herein. Preferably, said food or animal feed comprises phytaseas an additive which is active in the digestive tract, preferably thecrop and/or small intestine, of livestock, such as poultry and swine,and aquatic farm animals including fish and shrimp. Said additive isalso preferably active in food or feed processing.

In an alternative embodiment, phytase or phytase producing organisms areadded as a pretreatment to food or animal feed, such as in theprocessing of the food or feed. In this embodiment, the phytase isactive prior to consumption of the food or feed, but may or may not beactive at the time the food or animal feed is consumed.

Compositions comprising polypeptides or proteins possessing phytaseactivity may be prepared in accordance with methods known in the art andmay be in the form of a liquid or a dry composition. The polypeptide tobe included in the composition may be stabilized in accordance withmethods known in the art.

The invention additionally provides food or animal feed comprisingcells, spores or plant parts, including seeds, capable of expressingphytase as described herein.

Still further, the present invention contemplates a method for theproduction of a food or animal feed, characterized in that phytaseaccording to the invention is mixed with said food or animal feed. Saidphytase is added as a dry product or as a liquid, before or afterprocessing. According to one embodiment, wherein a dry powder is used,the enzyme is diluted as a liquid onto a dry carrier such as milledgrain.

Liquid compositions need not contain anything more than the phytaseenzyme, preferably in a purified form. Usually, however, a stabilizersuch as glycerol, sorbitol or mono propylene glycol is also added. Theliquid composition may also comprise one or more other additives, suchas salts, sugars, preservatives, pH-adjusting agents (i.e., bufferingagents), proteins, or phytate (a phytase substrate). Typical liquidcomposition are aqueous or oil-based slurries.

The liquid compositions can be added to a food or feed after an optionalpelleting thereof. Dry compositions may be spray-dried compositions, inwhich case the composition need not contain anything more than theenzyme in a dry form. Usually, however, dry compositions are so-calledgranulates which may readily be mixed with for example food or feedcomponents, or more preferably, form a component of a pre-mix. Theparticle size of the enzyme granulates preferably is compatible withthat of the other components of the mixture. This provides a safe andconvenient means of incorporating enzymes into for example an animalfeed.

Agglomeration granules are prepared using agglomeration techniques in ahigh shear mixer (e.g., Lodige) during which a filler material and theenzyme are co-agglomerated to form granules. Absorption granulates areprepared by having cores of a carrier material to adsorb/be coated bythe enzyme.

Typical filler materials are salts such as disodium sulphate. Otherfillers are kaolin, talc, magnesium aluminum silicate and cellulosefibers. Optionally, binders such as dextrins are also included inagglomeration granules.

Typical carrier materials are starch, e.g., in the form of cassaya,corn, potato, rice and wheat. Salts may also be used.

Optionally, the granulates are coated with a coating mixture. Suchmixture comprises coating agents, preferably hydrophobic coating agents,such as hydrogenated palm oil and beef tallow, and if desired otheradditives, such as calcium carbonate or kaolin.

Additionally, phytase compositions may contain other substituents suchas coloring agents, aroma compounds, stabilizers, vitamins, mineralsother feed or food enhancing enzymes and the like. This is so inparticular for the so-called pre-mixes.

A “feed” and a “food,” respectively, means any natural or artificialdiet, meal or the like or components of such meals intended or suitablefor being eaten, taken in, digested, by an animal and a human being,respectively.

A “food or feed additive” is an essentially pure compound or a multicomponent composition intended for or suitable for being added to foodor feed. It usually comprises one or more compounds such as vitamins,minerals or feed enhancing enzymes and suitable carriers and/orexcipients, and it is usually provided in a form that is suitable forbeing added to animal feed.

The phytases of the invention can also be used in poultry food toimprove egg shell quality (reduction of losses due to breaking), see forexample, The Merck Veterinary Manual (Seventh Edition, Merck & Co.,Inc., Rahway, N.J., USA, 1991, page 1268); Jeroch et al. Bodenkultur Vo.45(4):361-368 (1994); Poultry Science, 75(1):62-68 (1996); CanadianJournal of Animal Science 75(3):439-444 (1995); Poultry Science74(5):784-787 (1995) and Poultry Science 73(10):1590-1596 (1994).

An effective amount of the polypeptide in food or feed is typically fromabout 10 to 50,000 U/kg feed or food; preferably from about 10 to15,000, more preferably from about 10 to 10,000, in particular fromabout 100 to 5,000, especially from about 100 to about 2,000 U/kg feedor food.

The present invention also provides a method for the production of afood or animal feed, characterized in that cells, plant parts, includingseeds, and/or spores capable of expressing phytase according to theinvention are added to said food or animal feed. Such cells or spores,may be of any origin, bacterial, plant, or animal.

Further, the present invention provides for the use of the phytasedescribed herein with or without accessory phosphatases in theproduction of inositol and inorganic phosphate, and phytateintermediates.

Also provided is a method for the reduction of levels of phosphorous inanimal manure, characterized in that an animal is fed an animal feedaccording to the invention in an amount effective in converting phytatecontained in said animal feed.

In one embodiment, the transgene protein, for example phytase expressedin plants, especially seeds or grains, using the methods describedherein, is used in the production and synthesis of phytase. The phytasetransgene expressed by the recombinant nucleic acid of the invention maybe harvested at any point after expression of the protein has commenced.When harvesting from the seed or grain or other part of a plant forexample, it is not necessary for the seed or grain or other part of theplant to have undergone maturation prior to harvesting. For example,transgene expression may occur prior to seed or grain maturation or mayreach optimal levels prior to seed or grain maturation. The transgeneprotein may be isolated from the seeds or grain, if desired, byconventional protein purification methods. For example, the seed orgrain can be milled, then extracted with an aqueous or organicextraction medium, followed by purification of the extracted phytaseprotein. Alternatively, depending on the nature of the intended use, thetransgene protein may be partially purified, or the seed or grain may beused directly without purification of the transgene protein for food oranimal feed, food processing or other purposes.

Alpha-amylases break down starch 1-4 linkages. Amylases are enzymesfundamental to the brewing and baking industries. Amylases are requiredto break down starch in malting and in certain baking procedures carriedout in the absence of added sugars or other carbohydrates. Obtainingadequate activity of these enzymes is problematic especially in themalting industry. It has been known for some time that phytate has aninhibitory effect on amylases. A method of adequately increasing theactivity of amylases with a physiologically acceptable system, leads tomore rapid malting methods and, owing to increased sugar availability,to alcoholic beverages such as beers with reduced carbohydrate content.

Accordingly, seeds or grains with phytase expression provide advantagesin the production of malt and beverages produced by a fermentationprocess. Enhanced activity of amylases in grain increases the speed andefficiency of germination, important in malting, where malt is producedhaving increased enzymatic activity resulting in enhanced hydrolysis ofstarch to fermentable carbohydrates, thereby, improving the efficiencyof fermentation in the production of alcoholic beverages, for example,beer and scotch whiskey. Enhanced fermentation processes also find usein the production of alcohols that are not intended for humanconsumption, i.e., industrial alcohols.

The phytase and phytate-derived intermediates of the invention also finduse in many other agricultural, industrial, medical and nutritionalapplications. For example phytase and phytate-derived intermediates canbe used in grain wet milling. Phytate is used in cleaning products, rustremoval products and in the removal of metals and other polycations fromsuch diverse materials as waste products and carbonated beverages.Phytate and phytases may be used in the isolation and recovery of raremetals. Phytase may be used to produce lower phosphate homologs ofphytate, which may be used in dentifrice and other dental care productsas well as potential treatments or preventatives of bone resorption(e.g., in osteoporosis) and renal calculi (kidney stones). Phytate andderivatives have found use in the production of tofu, and chelation ofminerals (e.g., iron, zinc, calcium or magnesium) with phytate, followedby release with addition of phytase may provide a unique means ofproviding these nutrients. Phytases may be used in the production ofinositol from phytate its use in food products. Phytases may also beused in the chemical and biochemical synthesis of phosphate containingmaterials. Phytase, phytate and lower phosphate phytate derivatives findmany other uses in personal care products, medical products and food andnutritional products, as well as various industrial applications,particularly in the cleaning, textile, lithographic and chemical arts.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.The skilled artisan will appreciate that the methods disclosed may beapplied to any number of different species, including to obtain allsequences disclosed herein. All patent and literature references citedin the present specification are hereby incorporated by reference intheir entirety.

EXAMPLES Example 1 Preparation of Genomic DNA Encoding Phytases

Genomic DNA was prepared for several different microorganisms for thepurpose of undertaking a PCR reaction to determine whether phytases areencoded by the DNA for a particular organism.

Genomic DNA is obtained from Penicillium chrysogenum (deposit no. NRRL1951); Fusarium javanicum (deposit no. CBS 203.32); Fusariumvertisillibodes; Humicola grisea var. thermoidia deposit no. CBS 225.63or ATCC 22081; and Emericella desertorum deposit no. CBS 653.73 andisolated according to standard methods.

Alignments were performed for several known phytase sequences, includingthose from Aspergillus Niger/Aspergillus ficum, Aspergillus terreus 59,Aspergillus terreus 60, Aspergillus fumigatus, Aspergillus niger,Emericella nidulans, Talaromyces thermophylus and Myceliopthorathermophila. From these, several “boxes” were identified as beinglargely conserved, and from these primers were developed.

The following DNA primers were constructed for use in amplification ofphytase genes from the libraries constructed from the variousmicroorganisms. All symbols used herein for protein and DNA sequencescorrespond to IUPAC IUB Biochemical Nomenclature Commission codes. BOX1:primers coding for (V/L)L(A/S)RHGAR (SEQ ID NO:51) forward primerBTIYTIKCIMGICAYGGIHCIMG (SEQ ID NO:52) forward primerBTIYTIAGYMGICAYGGIHCIMG (SEQ ID NO:53) BOX2: primers coding forNNTL(D/E/H) (SEQ ID NO:54) forward primer AAYAAYACIYTISA (SEQ ID NO:55)reverse primer TSIARIGTRTTRTT (SEQ ID NO:56) BOX3: primers coding forLSPFC (SEQ ID NO:57) forward primer YTTTCICCITTYTGY (SEQ ID NO:58)forward primer YTIAGYCCITTYTGY (SEQ ID NO:59) reverse primerRCARAAIGGIGAIAR (SEQ ID NO:60) reverse primer RCARAAIGGRCTIAR (SEQ IDNO:61) BOX4: primers coding for G(N/S)PLGP (SEQ ID NO:62) forward primerGGIWVICCIYTIGGICC (SEQ ID NO:63) reverse primer CCIARIGGIBWICC (SEQ IDNO:64) BOX5: primers coding for DFSHD (SEQ ID NO:65) forward primerGAYTTYTCICAYGAY (SEQ ID NO:66) forward primer GAYTTYAGYCAYGAY (SEQ IDNO:67) reverse primer RTCRTGIGARAARTC (SEQ ID NO:68) reverse primerRTCRTGRCTRAARTC (SEQ ID NO:69) BOX6: primers coding for VR(A/V)I(I/V)NDR(SEQ ID NO:70) reverse primer CKRTCRTTIAYIARIRCICKIAC (SEQ ID NO:71)

Boxes were also developed according to the methods of Pasamontes et al.Appl. Evir. Microbial. 63(5):1696-1700 (1997) (expressly incorporatedherein) to provide the following primers. BOX2.5: coding for MDMCSFD(SEQ ID NO:72) forward primer ATGGAYATGTGYTCNTTYGA (SEQ ID NO:73) BOX4′:coding for YGHGAG (SEQ ID NO:74) reverse primer TTRCCRGCRCCRTGNCCRTA(SEQ ID NO:75)

PCR is performed on a standard PCR machine such as the PTC-150 MiniCycler from MJ Research Inc. (Watertown, Mass.) or an EppendorfMastercycler (Hamburg, Germany). In the experiments described below, PCRwas performed using a Hybaid Touchdown thermocycler (Middlesex, UK).

PCR conditions for Pwo polymerase (Boehringer Mannheim, Cat # 1644-947)comprise a 100 microliter solution made of 10 microliter of 10× reactionbuffer (10× reaction buffer comprising 100 mM Tris HCl, pH 8-8.5; 250 mMKCl; 50 mM (NH₄)₂SO₄; 20 mM MgSO₄); 0.2 mM each of dATP, dTTP, dGTP,dCTP (final concentration), 1 microliter of 100 nanogram/microlitergenomic DNA, 1 microliter of PWO at 1 unit per microliter, 500 mMprimers (final concentration) and water to 100 microliters. The solutionis overlaid with mineral oil.

Two approaches were developed for amplification of phytase genes fromthe genomic DNA:

A) A first PCR is run using BOX1 and BOX6 primers; the products are runon an agarose gel and approximately 1 kb fragments are isolated and runin a second PCR using nested primers. For the second PCR run, bestresults were obtained using primers from BOX1-BOX5 or from BOX5-BOX6 orBOX2.5/BOX4′.

Protocol A:

PCR1:

-   -   2′ at 94° C. (1 cycle)    -   45″ at 94° C.; 1′30″ at 40° C.; 1′30″ at 72° C. (30 cycles)    -   7′ at 72° C. (1 cycle)    -   hold at 4° C.

Fragments were put on a 1% low melting gel and fragments around theexpected size (0.0-1.2 kb) were sliced from the gel, isolated and usedas a template for the second PCR run (PCR2). PCR 2 followed the samecycling protocol as PCR1.

B) Touchdown PCR was performed using BOX2.5/BOX4′ primers. Using thistechnique, a specific fragment could be isolated, cloned into a TOPOvector (Invitrogen Corp., Carlsbad, Calif.), and sequenced withoutfurther processing.

Protocol B:

-   -   3′ at 95° C. (1 cycle)    -   1′ at 95° C.; 1′ at 60° C., decreasing to 50° C.; 30″ at 72° C.        (20 cycles, so that the temperature dropped 0.5° C. each cycle        in the annealing step)    -   1′ at 95° C.; 1′ at 50° C.; 30″ at 72° C. (10 cycles)    -   hold at 4° C.

From the sequenced fragments, it was possible to use the RAGE technique(rapid amplification of genomic ends) to rapidly obtain the sequence ofthe full length gene. Using the GenomeWalker™ Kit from ClontechLaboratories, Inc (Palo Alto, Calif.) and manufacturer's protocol(GenomeWalker™ Kits User Manual, published Nov. 10, 1999, expresslyincorporated herein), adapter ligations were derived from the fragmentsequences to further determine upstream gene sequence. Sequences ofphytase genes were determined from chromosomal DNA of various species.

FIG. 22 shows the phytase polynucleotide sequence of E desertorum (SEQID NO:47) obtained by the above methods and the sequences therein fromwhich adapter ligations (primers GSP1 rev:fyt037 and GSP2rev:fyt036)were derived to obtain the upstream sequences encoding this phytase (seeFIG. 17A).

FIG. 23 shows the phytase sequence of F. javanicum (SEQ ID NO:49)obtained by the above methods and the sequences therein from whichadapter ligations (primers GSP1 rev:fyt039 and GSP2rev:fyt038) werederived to obtain the upstream sequences encoding this phytase (seeFIGS. 18A-18B).

Example 5 Evidence of Phytate Hydrolyzing Activity in Liquid Culture

A selected fungal species is grown in defined media containing variousconcentrations of inorganic phosphate, and growth characteristics andphytase production are assayed and compared. Spore suspensions are used(2×10⁶ spores/ml final con) to inoculate a minimal media (Vogels) wherethe phosphate concentration is altered to see how this will affectgrowth and phytase production. Cultures are grown in 50 ml of medium inshake flask culture at 25° C. to 30° C. Cultures are harvested at 24,48, 72 and 96 hours. Culture supernatants are assayed for phytaseactivity using the method of Fiske and SubbaRow, Journal of BiologicalChemistry 66:375-392 (1925). Growth may be determined: by dry weight orOD readings.

5A. Effect of Different Media Conditions on Growth and Morphology

A series of fungal growth curves are produced to look at the effect ofavailable P in the medium on growth and phytase production. In someinstances, when the P level is reduced, morphological changes in thegrowth of the fungus are observed which are associated with a stressedcondition (e.g., mycelial fragmentation, pelleting, heterogeneous growthand an overall appearance of a pale yellow color). This physiologicalstrain may be related to the appearance of phytase activity at a pointin the growth curve, for example approaching late exponential phase.Morphological evidence of phytic acid utilization may be observed incultures of low P (e.g., 0.57 mM) supplemented after 24 hours growthwith 1 mM phytate as a phosphorus source. The morphological changes seenwithout added phytate may not be apparent, indeed the supplement samplesmay resemble cultures in media of higher P which were not limiting. Thisresponse would indicate that a phytic acid specific hydrolyzing activitywas being produced so that P could be supplied to the growing fungus. Asa caveat, it is possible that higher concentrations of phytate (e.g., 5mM) supplementing the cultures result in a lack of cell growth. Such aresult would suggest that the high level of phytate in the mediumchelates essential minerals resulting in a medium that cannot supportfungal growth and nutrition.

In an exemplary study, the fungus is grown in media containing

-   -   High phosphate (1.14 mM)    -   Low phosphate (0.57 mM)    -   Low phosphate plus 1 mM supplemented phytate.

Growth is monitored over 0, 24, 48, 72 and 96 hours by dry weightmeasurements, and the morphological characteristics in response to thedifferent media conditions are also observed. In a situation wherephytate hydrolyzing activity which allows the fungus to access phosphatefrom phytate, and so circumvent phosphate starvation stresses that theculture may otherwise experience, the major observations that would beexpected are:

-   -   1. Good growth in high phosphate, consistent fungal morphology        indicative of healthy culture.    -   2. Markedly poorer growth in low phosphate condition, fungal        morphology heterogenous with evidence of clumping and mycelial        fragmentation. The culture may have a sickly yellow appearance.    -   3. Similar cultures as for (2), when supplemented with phytate        (the substrate), no longer appear to be under the same        physiological stress. Biomass growth is similar to condition (1)        and the fungal morphology is the same as for the high phosphate        condition.    -   4. Growth curves and photographic evidence support these        observations.

5B. Phytase Activity in Culture Supernatants

Phytase activity in the supernatants of fungi growing on media withvariable levels of inorganic P can be measured. Supernatant samples areused to compare activities at a specified time post inoculation. Phytaseactivity may be expressed as the number of mmoles P released per minuteper ml culture supernatant. Sample activities are calculated fromtriplicate culture flasks where supernatants are assayed for phytase induplicate. Activities are shown as mean_SD. Along with the observationsabove, a clear physiological stress associated with cultures wherephosphate is limited, which adversely affected growth, may be observedand linked to the appearance of phytase activity.

5C. Concentration of Culture Supernatants

Additional evidence of phytase activity can be expected fromconcentrated supernatant (concentrated protein). For example,concentrated protein samples can be obtained from:

-   -   1. Cultures of fungus from conditions of stress and low        phosphate (where phytase is expected to be expressed),    -   2. Cultures of fungus of high phosphate and no stress, where        phytase is not expected to be produced, and    -   3. Cultures supplemented with low phosphate and supplemental        phytate.

Silver stained SDS-PAGE gels of these concentrated protein samples areexpected to show a protein profile demonstrating the appearance of aprotein band (putative phytase band) in concentrated protein fromcondition 1 (above) which is not present in condition 2. A similarappearance of this band is also expected in condition 3, albeit at alower level. Based on the amino acid sequence of a specific phytase, andon whether it appears to be an extracellular enzyme, the size of theprotein may be approximated. It should be noted, however, thatglycosylation modification on the extracellular enzyme may increase theMW.

Example 6 PCR Amplification of Phytase Gene Fragments

6A Degenerate Primer Design

Based on alignments of published phytase amino acid sequences, a rangeof degenerate primers are designed against conserved structural andcatalytic regions. Such regions included those that are highly conservedamong the phytases, as well as those known to be important for enzymestructure and function.

For example, amino acid sequences for published phytases are aligned. Itshould be noted that many phytase sequences are publicly available fromGenBank, and each is incorporated herein by reference.

Particular regions are chosen to meet the criteria above, and a range offorward and reverse primers designed from the amino acid sequences.Using the genetic code for codon usage, degenerate nucleotide PCRprimers are synthesized.

As another example, primers are designed from the published amino acidsequence for different phytases from a single species (e.g., A. niger).These primers may be designed as follows:

-   -   1. Primer 1: Forward (5′-3′) primer from, for example, the        phosphate binding domain of a phytase, which should be essential        for catalytic activity.    -   2. Primer 2: Reverse primer from a central phytase region which        seems to be conserved relatively well.

All primers may be synthesized in the 5′-3′ direction. The standardgenetic code is used to change from amino acid to triplet codon, andstandard IUB code for mixed base sites are used (e.g. to designate I forA/C/T/G).

As can be seen from the alignment of sequences for A. niger PhyA andPhyB, the phosphate-binding domain is well conserved with only a singleamino acid difference between PhyA (RHGARYP; van Hartingsveldt et al.,1993, SEQ ID NO:76) and PhyB (RHGERYP; Piddington et al., 1993, SEQ IDNO:77). A degenerate primer may be designed complementary to this regionin the PhyA version of the sequence only, i.e. using RHGARYPT (SEQ IDNO:78) as the basis for primer design. This would be to bias the primertowards a PhyA type phosphate binding domain. A second conserved region,which may serve as the basis for primer 2 for A. niger-derived primers,occurs in the middle of the PhyA and PhyB amino acid sequence. Thisconserved central phytase-specific domain in PhyA (FTHDEWI, SEQ IDNO:79) corresponds to amino acids 285-291. In PhyB, the amino acidsequence (FTQDEWV, SEQ ID NO:80) corresponds to amino acids 280-286.

Degenerate primers developed as described above may be used to amplify aphytase encoding region from other species by PCR, as described next.

6B. PCR amplification of Phytase Gene Fragments

Genomic DNA from a species of interest may be used as a template for PCRamplification of putative phytase gene fragments using combinations ofprimers made as described above. PCR is carried out using the PCRReady-to-go Beads from Amersham Pharmacia. Conditions are determined byindividual experiments, but typically thirty cycles are run in a Technethermal cycler. Successful amplification is verified by electrophoresisof the PCR reaction on a 1% agarose gel. A PCR phytase product that isamplified by the primers may be anticipated by a correct expected size.The product is then purified by gel extraction using the Qiaquick SpinGel Extraction kit from Qiagen. The purified PCR product is ligated intothe commercial pGEM-T Easy vector System (Promega Corporation) tofacilitate cloning. Ligation reactions are incubated at 4° C. overnightin a total volume of 10 ml containing 0.1 volumes of 10× ligase bufferand 1 ml (1 U.ml⁻) of T4 DNA ligase. Typically insert DNA is used in thereaction in a 1-4:1 molar ratio of insert to vector DNA. A 100 mlaliquot of CaCl₂ competent E. coli XL-1 Blue cells are removed from −80°C. storage and thawed on ice for transformation. 3 ml of ligation mix isadded to the cells and the mixture incubated on ice for 20 min. Thecells are then heat shocked at 42° C. for 1 min. and returned to ice for5 min. The transformation mixture is added to 0.9 mL of L-broth, and thecells incubated with shaking and without selection to allow expressionof the ampicillin resistance gene product before selection is applied(37° C., 1 h). Aliquots of 200, 300 and 400 ml of this culture are thenspread directly on selective agar plates. Plates are incubated at 37° C.overnight. Colonies containing recombinant plasmids are visualized usingblue/white selection. For rapid screening of recombinant transformants,plasmid DNA is prepared from cultures of putative positive (white)colonies. DNA is isolated by the method of Birnboim and Doly followingthe protocol in Sambrook et al. (1989). The presence of the correctinsert (650 bp) in the recombinant plasmid is confirmed by restrictionanalysis. DNA is digested with restriction enzymes (e.g., Not1-pPst1)overnight at 37° C., and digest products visualized by agarose gelelectrophoresis. A number of clones may contain the correct sized insertand can be selected for manual sequencing to see if the insert is aphytase gene fragment. Inserts are sequenced using the dideoxy chaintermination method of Sanger et al (1977) with a modified form of T7 DNApolymerase (Sequenase version 2.0). The reactions are carried out usingreagents supplied in the Sequenase version 2;0 kit (Amersham LifeScience-United States Biochemical Corporation), following themanufacturer's protocol. Partial sequence from the ends clones mayindicate that a phytase gene fragment had been cloned. Full sequencingof the double-stranded inserts is performed on plasmid DNA from theseclones.

6C. Sequence Analysis

The sequences are analyzed by BLAST and protein translation sequencetools. BLAST comparison at the nucleotide level may show various levelsof homology to published phytase sequences. Initially, nucleotidesequences are submitted to BLAST (Basic BLAST version 2.0) by accessingthe BLAST database on the world wide web. The web site used is atncbi.nlm.nih.gov/cgi-bin/BLAST. The program chosen is blastn, and thedatabase chosen is nr. Standard/default parameter values are employed.Sequence data for putative gene fragments are entered as sequence inFASTA format and the query submitted to BLAST to compare these sequencesto those already in the database.

The sequences are then subjected to a DNA-to-protein translation toolcalled Protein machine. This tool is also available on the web atmedkem.gu.se/edu/translat.html. Another suitable translation tool isknown as Translation Machine, available on the web atwww2.ebi.ac.uk/translate/. The DNA sequences of putative phytase genefragments are inserted into the analysis block, and the standard geneticcode is used as the basis for the translation. Translations are carriedout in all three frames and on forward and reverse strands. Thetranslated amino acid sequence is delivered on the screen by theanalysis tool as amino acid sequence in one letter code. Ideally,analysis of the amino acid sequence will show that the fragment containsboth correct ends (as used to design the primers), contains theessential P binding motif and perhaps other residues which are alsopresent in published phytase sequences. From this, it may be concludedthat the fragment cloned is a phytase gene fragment.

Sequence alignments and analysis of those alignments is carried out atthe nucleotide and amino acid level using the ALIGN program (AlignmentEditor Version 4/97; Dominick Hepperle, Fontanestr. 9c, D016775,Neuglobsow, Germany). In performing the analysis, subject sequences arepasted in, and the PHYLIP Interleaved format employed. The homologyanalysis is carried out using the “Analyze” section of the program, andspecifically the option entitled “Distance Analysis.” This calculates %homologies and the number of different sites between species, using aminimum of two amino acid sequences (i.e., two “species”). Minimal andmaximal homologies are calculated as %. The basis for homology analysisis done as % identity, on the calculation of “number of identical aminoacids (or bases) divided by the total number of amino acids (or bases)multiplied by 100” to give a percentage value. Amino acid sequences areplaced into the ALIGN program along with published phytase sequences anda manual alignment at the amino acid level is carried out. From this,the deduced translation for the PCR product obtained using degenerateprimers may be obtained.

Example 7 Southern Analysis for Library Production

Genomic DNA from different species is digested with a range ofrestriction enzymes overnight at 37° C. Successfully digested DNA is runout on a 1% agarose gel in preparation for transfer to the nylonmembrane. After completion of electrophoresis, the agarose gel is soakedfor 10 min. in 0.2M HCl to depurinate the DNA and then rinsed briefly inddH₂O. The DNA is transferred to the Hybond-N+ membrane (AmershamInternational PLC) by alkali capillary blotting. The blot is set up sothat the nylon filter is sandwiched between the gel and a stack ofabsorbent paper towels. A wick of Whatman 3MM paper (Schleicher andSchuell, Dassel, Germany) is prepared on a glass plate over a reservoirof transfer buffer (0.4M NaOH). The gel is inverted on the wick, takingcare to avoid the formation of air bubbles, and surrounded by strips ofNescofilm to prevent the blotting action of the paper towels fromby-passing the gel at its edges. The gel is covered with an equal sizedpiece of Hybond-N+ membrane which had been cut in the corner to matchthe gel and pre-wetted in 3×SSC. Next, 3-5 pieces of 3MM paper areplaced on top of the filter and the blot completed by adding a 10 cmstack of blotting paper followed by a 0.5 kg weight. The blot is leftfor 8-24 h to transfer the DNA. The membrane is then washed briefly in2×SSC at RT and baked in a vacuum oven at 80° C. to fix the DNA to themembrane. An isolated fragment from the procedures above is used toprobe the Southern blot. It is firstly labeled with ³²P isotope by useof the High Prime DNA Labeling Kit (Boehringer Mannheim). Denaturedfragment is added into a random primed labeling reaction whichincorporates radio-labeled adenine. The Southern blot is prehybridisedfor 1 hour at 42° C. in 12 mL of Easy-Hyb buffer (Boehringer Mannheim)in a hybridization tube. Radiolabeled probe is denatured and added to 5mL of Easy-Hyb hybridization buffer and left to hybridize overnight at42° C. Following hybridization, the blot is washed by incubation in 40mL 3×SSC, 0.1% SDS for 15 min at 42° C. This low stringency wash isrepeated with fresh wash solution. After stringency washing, the lot isrinsed in 3×SSC, sealed in clear plastic and exposed to x-ray film. Thisis left for 2 hours and the film developed.

Strong hybridizing bands may be observed for a given species digest.Such results indicate that the fragment can be used as a probe forlibrary screening.

Example 8 Isolation of a Polynucleotide Sequence from the Genome of aSpecies of Interest Encoding a Phytase

8A. Genomic Library Generation and Screening

Following the Southern hybridization analysis, a partial genomic librarymay be made in order to try and clone a full-length phytase gene. A sizerestricted plasmid library targeting a digestion fragment (as estimatedfrom Southern analysis) is generated. Digested genomic DNA is run out ona 1.25% agarose gel. The digested fragments of a preferred approximatesize are extracted from the gel, and purified by Glass-Max (Gibco-BRL,Scotland). Purified genomic fragments are used in a shotgun ligationreaction with restriction nuclease linearized pSK II Bluescript vector(Stratagene). The vector is first dephosphorylated before ligation, andthe ligation reaction is carried out at 14° C. overnight. The library isproduced by transformation of E. coli XL-10 Gold ultracompetent cells(Stratagene). 100 ml aliquots cells are removed from −80° C. storage andthawed on ice for transformation. 4 mL of b-mercaptoethanol is added tothe cells on ice. 3 ml of ligation mix is added to the mixture and themixture incubated on ice for 20 min. The cells are then heat shocked at42° C. for 30 sec and returned to ice for 2 min. The transformationmixture is added to 0.9 mL of NZY-broth, and the cells incubated withshaking and without selection to allow expression of the ampicillinresistance gene. The transformed cells are plated out on blue/whiteselection LB-agar plates, and left to incubate overnight at 37° C. Thecolonies are lifted onto nitrocellulose filters by the method ofManiatis (10% SDS—lysis, 3 min; 1.5M NaOH-denaturation, 5 min; 1.5MTricHCl—neutralisation, 5 min; 3×SSC—rinse, 5 min). The filters are thenbaked for 2 hours at 80° C. under vacuum to fix the DNA. The library isscreened with ³²P radiolabeled 636 bp probe in the same manner as forSouthern hybridization. After hybridization the filters are washed twicein 3×SSC, 0.1% SDS, 42° C., 15 min. The filters are then rinsed in3×SSC, sealed in plastic and exposed to X-ray film overnight at −80° C.Positive hybridizing spots are identified on the film. These are alignedto the agar plates containing the transformants. The hybridizing spotsare matched up single colonies on the agar plates. All colonies in theradius of the hybridizing spot are picked up using sterile loops andused to inoculate 2 mL of Luria broth. The cultures are grown at 37° C.for 2 hours. Dilutions of the cultures are made from 10⁻¹ to 10⁻⁵ and100 mL of each sample is plated out on LB-amp agar plates and incubatedovernight at 37° C. The plates which have between 10 and 150 colonies onthem are chosen to go forward for a secondary screen. Colony lifts aredone as before, and filters are processed using the same procedures.Fresh ³²P labeled probe is prepared, and the filters screened in thesame way as outlined previously. Stringency washes are carried out using2×SSC, 0.1% SDS at 42° C. for 15 min. Filters are then rinsed in 2×SSC,sealed in plastic and exposed to X-ray film for 2 hours. The developedfilm should shows hybridizing spots, consistent with amplification ofthe positive colonies from the primary screen. The film is then alignedto the plates, and the spots co-ordinated to see if they corresponded tosingle isolated colonies. The best positives that match up to singlecolonies are picked and used to inoculate Luria broth for plasmid DNApreparations. Plasmid DNA is purified by Qiaspin Mini-Prep kit (Qiagen)and restriction analysis carried out to estimate the size of theinserts. All clones giving the same restriction profile can be used tosuggest an insert size. Clones may be partially sequenced to determineif they are the correct gene/gene fragment. The full sequence of theseclones is then determined.

8B. Percentage Identity Comparison Between Fungal Phytases

The deduced polypeptide product of the cloned phytase gene fragment isused for homology analysis with published phytases. The analysis showspercent identities and, together with analysis of the translatedsequence, may provided evidence that the gene fragment cloned is ahomolog of a specific phytase.

8C. Generation and Screening of a Restriction Enzyme-BasedSize-Restricted Genomic Library to Isolate Remainder of Phytase Gene

In order to isolate the remaining portion of a gene, a secondrestriction enzyme may be used to generate a second partial genomiclibrary, and fragments may then be subcloned together. The restrictionendonuclease recognition sites present within a cloned phytase sequenceare identified using Webcutter. Of particular interest are sites forenzymes that are used in the Southern analysis discussed above. Verylarge fragments (e.g., 8 Kb), would be difficult to clone in aplasmid-based library a low degree of hybridization with a specificrestriction enzyme band argues against use of such in a library screen,and the presence of two bands in a restriction enzyme lane is likely tocomplicate the screening process. The library is made as before inpBluecript SKII, and screened using the same probe. A selection ofpositive hybridizing colonies are chosen and aligned to colonies on theplates. Matching colonies are picked for plasmid DNA preparations.Restriction analysis may show how many clones have inserts. These clonesare then fully sequenced.

8D. Amplification of Contiguous Phytase Gene for Heterologous Expression

A composite phytase sequence is produced from genomic clones and used todesign a number of upstream and downstream primers which could be usedto amplify a contiguous phytase gene sequence. PCR amplification is alsodesigned to facilitate cloning and expression of the complete phytasegene in to a heterologous expression vector (e.g., pGAPT-PG, a 5.1 Kbconstruct provided by Genencor International, Inc.). Restriction enzymesites within the multiple cloning site of the vector which are notpresent within the phytase gene sequence are determined. A number of 5′and 3′ flanking primers may be designed using the phytase gene sequence,and modified to include the restriction enzyme recognition sites forthese enzymes.

Restriction enzyme recognition sites are designed into the primersequences to facilitate cloning into the expression vector. The upstreamand downstream flanking regions used to design the primers arearbitrarily chosen at approximately 100 bp upstream from the ATG (start)codon and downstream from the TAG (stop) codon respectively. The genesequence used is also chosen to contain as equal balance of bases aspossible.

Amplification of the phytase gene by PCR may be done using genomic DNAcombinations of primers. PCR should amplify a region corresponding tothe full-length phytase gene. The desired product produced byamplification with the primers is cloned into a vector and severalclones which contain the correct size of insert are selected forsequencing. Homology analysis of the clone sequences is then performedand a full length phytase sequence determined.

PCR amplification genomic DNA is carried out using a combination of 5′primers and 3′ primers, and using a high fidelity DNA polymerase, Pfu,to minimize error for expression of the phytase gene. This polymerase isPfu DNA polymerase (Stratagene) and comes as part of the Pfu DNApolymerase kit for PCR. For these reactions, reaction buffer, dNTPs,target DNA and primers are mixed together, and 2.5 units of Pfupolymerase added in a final reaction volume of 50 μL. Afteramplification, a 5 μL aliquot of the reaction mixture is analyzed by gelelectrophoresis. Selected fragments are cloned directly into the vectorpCR-Blunt II TOPO (Invitrogen), and a select number of clones analyzedto confirm the presence of the correct insert. (Blunt-ended PCR productsthat are generated by Pfu DNA polymerase are cloned into the ZeroBlunt®TOPO®PCR cloning kit (Invitrogen). This vector contains a MCS siteand a kanamycin gene for antibiotic resistance, but also allowsselection based on disruption of the lethal E. coli gene ccdb, asopposed to blue-white selection. Purified PCR product (50-200 ng) isadded to 1 μL of pCR-BluntII-TOPO vector and the reaction volume made upto 5 μL with sterile water. This is mixed gently and left to incubatefor 5 min at room temperature. 1 μL of 6×TOPO Cloning Stop Solution isadded, and the reaction left on ice or frozen at −20° C. for up to 24hours for transformation.) The integrity of the engineered restrictionsites are also confirmed by this analysis. A number of clones areprepared and sequenced. Sequence analysis may confirm the presence of afull-length phytase gene. This gene may then be taken forward forexpression in a heterologous system, and subsequent biochemicalcharacterisation of the enzyme.

8E. Analysis of Phytase Sequence

An alignment is made of the isolated sequence and published phytases andhomology analysis done, on a % identity basis.

Example 9 Cloning, Expression and Characterization of the Phytase

Over-expression of the phytase gene in a heterologous host may be doneto produce enough protein to carry out characterization of the enzyme.

9A. Cloning of Phytase Gene Into Expression Vector and Transformation into a Host

The full-length phytase gene is amplified with a high-fidelity DNApolymerase, is produced using primers that are engineered to contain tworestriction enzyme sites (e.g., EcoRV and AgeI). These sites are used tofacilitate cloning into the expression vector (e.g., pGAPT-PG). Thephytase clones are digested with the enzymes to produce a single insertfragment. The vector is also digested with these enzymes and linearize.The phytase gene fragment is ligated to the expression vector, and anumber of transformants produced. A selection of these clones isanalyzed to confirm the presence of the insert. The phytase clones arethen used to transform swollen spores of A. nidulans by electroporation.

The transformation of host such as A. niger strain FGSC A767 and A.nidulans FGSC A1032 by electroporation is adapted from the protocol ofO, Sanchez and J. Aguirre developed for A. nidulans. 50 mL of YG medium(0.5% yeast extract, 2% glucose, supplemented with 10 mM uridine and 10mM uracil) is inoculated at 10⁷ spores/mL with appropriate sporesuspension. The cultures are grown for 4 hr at 25° C. at 300 rpm onrotary shaker. Swollen spores are collected by centrifugation at 400 rpmfor 5 min at 4° C. Spores are resuspended in 200 mL ice-cold sterilewater and centrifuged at 4000 rpm for 5 min at 4° C. The supernatant ispoured off and the spores are resuspended in 12.5 ml YED media pH 8.0(1% yeast extract, 1% glucose, 20 mM HEPES) and incubated for 60 min at30° C. at 100 rpm on rotary shaker. The spores are collected bycentrifugation at 400 rpm for 5 min, then resuspended in 1 mL ofice-cold EB buffer (10 mM tris-HCl, pH 7.5, 270 mM sucrose, 1 mM Lithiumacetate) at a concentration of 10⁹ conidia.mL⁻¹ and kept on ice. 50 μlof the swollen spore suspension is mixed with 1 to 2 μg DNA in a totalvolume of 60 μl in sterile Eppendorf and kept on ice for 15 min. Thesuspension is transferred to 0.2 cm electroporation cuvette.Electroporation is carried out in a BioRad electroporation device(settings 1 kV, 400 W, 25 μF). 1 mL of ice-cold YED is added to thesuspension after electroporation, and the combined mix is transferred toa pre-chilled sterile 15 mL Falcon tube and kept on ice for 15 min. Thisis then incubate at 30° C. for 90 min at 100 rpm on rotary shaker, withthe tubes in a horizontal position. The spores are plated out andtransformants are observed after 36-48 hours.

Circular plasmid DNA may be used. A. niger strain FGSC A767 and A.nidulans strain FGSC A1032 can be obtained from the Fungal GeneticsStock Center, University of Kansas Medical Center, 3901 RainbowBoulevard, Kansas City, Kans., USA.

9B. Preliminary Characterization of Transformants

Transformants are selected for further analysis. Spores from each ofthese transformants are used to inoculate selective media, and sporesuspensions of each clone are made. These are used to inoculate liquidcultures of the transformants which are screened for phytase activity.Cultures are grown over 72 hours, and the supernatants collected.Samples are desalted in PD-10 columns, and the protein samples eluted in0.25 M sodium acetate. Phytase assays are carried out in the standardconditions (pH 5.5, 37° C. for 30 min). Clones are identified havingphytase activity. These are taken forward for further analysis.

9C. Time of Maximal Expression of Phytase in Liquid Culture

In order to assess when the level of phytase production is at itshighest for subsequent biochemical characterisation, a series of liquidcultures of clones are generated over a 2-day to 7-day period. Culturesare inoculated with spore suspension of the appropriate transformants,and harvested at each day over this period. Culture supernatants areprocessed as standard, and the desalted culture supernatant is assayedunder standard phytase conditions. The time point of highest phytaseactivity is then determined

Liquid cultures are harvested at each time point, desalted and eluted in0.25 mM sodium acetate pH 5.5. Phytase assays are carried out understandard conditions (pH 5.5, 37° C., 30 min) in duplicate. Activity isexpressed in phytase units per mL of culture supernatant (μmoles of Pireleased min-1 mL-1).

Untransformed host may also be assayed across these time-points as acontrol. Protein samples from selected supernatant samples (day 4 andday 6), both before and after desalting are analyzed by SDS-PAGE todetermine levels of secretion.

9D. Southern Analysis of Transformants

Although there may be evidence that the phytase gene has beensuccessfully cloned into the expression vector, and that expression ofan active enzyme had been achieved, molecular evidence may also beobtained. Genomic DNA preparations are made from the transformed host,and from the original untransformed host. The DNA is digested with arestriction enzyme, preferably one where there is no internal sitewithin the phytase gene, and Southern hybridization analysis of thetransformants is carried out. The Southern blots are analyzed with aphytase probe from species under investigation. Single stronghybridizing bands seen for the transformants under conditions of mediumto high stringency (3×SSC) indicate successful cloning. If there is noevidence of any other hybridizing bands, it can be concluded that asingle-copy of the phytase gene is present in the transformed host. Alack of hybridizing bands in the untransformed sample indicates thatthere is no homology between the phytase of interest and any phytasespresent in the host genome.

9E. Biochemical Characterization of a Phytase

To prove that the cloned gene represents a specific phytase activity,and to characterize that activity, a range of biochemical analyses arecarried out on the over-expressed enzyme. Preliminary characterizationmay indicate that the gene is producing a phytic-acid hydrolyzingactivity. This analysis can be extended to examine activity at differentpHs, temperatures and against different substrates.

Transformants are taken forward for these analyses, and cultures areharvested at optimum expression time, as determined above. With phyticacid as the substrate, the pH effect on enzyme activity can be shown.The purified enzyme sample is desalted from culture supernatant, andeluted in 0.025 mM sodium acetate pH 5.0. This is then added tosubstrate which is made in solutions of the following buffers: pH3.0:0.4M glycine-HCl, pH 4.0: 0.4M Sodium acetate, pH 5.0:0.4M Sodiumacetate, pH 6.0:0.4M imidazole-HCl, pH 7.0:0.4M Tris-HCl, pH 8.0:0.4MTris-HCl pH 9.0:0.4M Tris-HCl. An optimum pH for the phytase activitymay be determined, as well. Little activity seen when4-nitrophenyl-phosphate is used as the substrate indicates a high levelof specificity for the phytic-acid substrate.

The temperature profile of the enzyme is characterized using pH 5.0buffer, over a range of temperatures, using phytic acid as thesubstrate. The phytase temperature activity range and optimum activitytemperature can be determined.

Preliminary stability studies may also be carried out on the phytase.Samples of the protein are left at −20° C., 4° C., and 37° C. overnightand then assayed under standard conditions. Samples may also be exposedto high temperature (e.g., 80-105° C. for 5-25 minutes) to determine thethermostability of the phytase activity. Residual activity is based oncomparison to phytase activity determinations taken from the samplesbefore exposure to each condition. Samples may be assayed afterwards inthe same assay conditions.

Of course, it should be understood that a wide range of changes andmodifications can be made to the preferred embodiment described above.It is therefore intended that the foregoing detailed description beunderstood in the context of the following claims, including allequivalents, which are intended to define the scope of this invention.

The Sequence Listing is contained on a separately submitted CD-ROMentitled GC645-2-US-seqlist.TXT (151 KB) created Sep. 15, 2004 which isincorporated in its entirety by reference herewith.

1-12. (canceled)
 13. An isolated polynucleotide encoding an enzyme, saidenzyme an amino acid sequence having at least 55% identity to an aminoacid sequence as disclosed in FIG. 8 or 21 (SEQ ID NO:8).
 14. Anisolated polynucleotide including a nucleotide sequence (i) having atleast 55% identity to a nucleotide sequence as disclosed in FIG. 7, and21 (SEQ ID NO:7), or (ii) being capable of hybridizing to a probederived from the nucleotide sequence disclosed in FIGS. 7 and 21 (SEQ IDNO:7) under conditions of intermediate to high stringency, or (iii)being complementary to a nucleotide sequence having at least 55%identity to a nucleotide sequence as disclosed in FIG. 7 and 21 (SEQ IDNO:7).
 15. An expression construct including a polynucleotide accordingto claim
 13. 16. A vector including the expression construct of claim15.
 17. A host cell transformed with the vector of claim
 16. 18. Food oranimal feed including an enzyme having phytase activity, wherein saidenzyme includes an amino acid sequence having at least 55% identity toan amino acid sequence as disclosed in FIG. 8 or 21 (SEQ ID NO:8).
 19. Amethod of producing an enzyme having phytase activity, comprising: (a)providing a host cell transformed with an expression vector comprising apolynucleotide as defined in claim 13; (b) cultivating said transformedhost cell under conditions suitable for said host cell to produce saidphytase; and (c) recovering said phytase.
 20. The method of claim 19,wherein said host cell is an Aspergillus species.
 21. A purified enzymehaving phytase activity, produced by the method of claim
 20. 22. Amethod of separating phosphorous from phytate, comprising: treating saidphytate with an enzyme (i) having phytate hydrolyzing activity and (ii)including an amino acid sequence having at least 55% identity to anamino acid sequence as disclosed in FIG. 8 or 21 (SEQ ID NO:8).
 23. Anisolated polynucleotide encoding an enzyme having phytase activity,wherein said enzyme includes an amino acid sequence having at least 55%amino acid sequence identity to SEQ ID NO:8.
 24. Food or animal feedincluding an enzyme having phytase activity, wherein said enzymecomprises an amino acid sequence having at least 55% amino acid sequenceidentity to SEQ ID NO:8.
 25. The method according to claim 19, whereinthe polynucleotide encodes an enzyme having phytase activity and atleast 55% sequence identity to SEQ ID NO:8.
 26. An expression constructincluding a polynucleotide according to claim
 14. 27. A vector includingthe expression construct of claim
 26. 28. A host cell transformed withthe vector of claim
 27. 29. A method of producing an enzyme havingphytase activity, comprising: (a) providing a host cell transformed withan expression vector comprising a polynucleotide as defined in claim 14;(b) cultivating said transformed host cell under conditions suitable forsaid host cell to produce said phytase; and (c) recovering said phytase.30. The method of claim 29, wherein said host cell is an Aspergillusspecies.
 31. A purified enzyme having phytase activity, produced by themethod of claim 30.